Methods and compositions for rapid plant transformation

ABSTRACT

The disclosure pertains to methods and compositions for the rapid and efficient transformation of plants. The disclosure further provides methods for producing a transgenic plant, comprising (a) transforming a cell of an explant with an expression construct comprising (i) a nucleotide sequence encoding a WUS/WOX homeobox polypeptide; (ii) a nucleotide sequence encoding a polypeptide comprising two AP2-DNA binding domains; or (iii) a combination of (i) and (ii); and (b) allowing expression of the polypeptide of (a) in each transformed cell to form a regenerable plant structure in the absence of exogenous cytokinin, wherein no callus is formed; and (c) germinating the regenerable plant structure to form the transgenic plant. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present disclosure.

FIELD OF THE DISCLOSURE

The present disclosure relates generally to the field of plant molecularbiology, including genetic manipulation of plants. More specifically,the present disclosure pertains to rapid, high efficiency methods andcompositions for producing a transformed plant in the absence ofcytokinin and without callus formation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/249,318, filed Aug. 26, 2016, which claims the benefit of U.S.Provisional Application No. 62/248,578, filed Oct. 30, 2015, which ishereby incorporated herein in its entirety by reference.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named20220107_6752-US-CNT_SeqList.txt, created on Jan. 7, 2022, and having asize of 1,168,973 bytes and is filed concurrently with thespecification. The sequence listing contained in this ASCII formatteddocument is part of the specification and is herein incorporated byreference in its entirety.

BACKGROUND

Major advances in plant transformation have occurred over the lastseveral years. Transformation of a variety of agronomically importantplants, e.g., maize, soybean, canola, wheat, indica rice, sugarcane andsorghum, and inbred lines continues to be both difficult and timeconsuming. Traditionally, the only way to elicit a culture response hasbeen by optimizing media components and/or explant material and source.This has led to success in some genotypes, but many important cropplants, including elite inbreds or varieties, fail to produce afavorable culture response. Although transformation of model genotypescan be efficient, the process of introgressing transgenes intoproduction inbreds is laborious, expensive and time consuming. It wouldsave considerable time and money if genes could be introduced into andevaluated with greater speed and efficiency.

Despite limitations, Agrobacterium-mediated transformation of monocotssuch as corn, rice, and wheat remains a widely used experimentalapproach, often with the use of meristematic tissue such as immatureembryos as the explants of choice (e.g., Ishida et al., 1996; Zhao etal., 2001; Frame et al., 2002). For rice, transformation of imbibedseeds has also been reported (Toki et al., 2006). To date, the mostcommon methods used for contacting cells with Agrobacterium include:culturing explant tissue such as immature embryos (“co-culture”),possibly including a “delay” or “resting” (non-selective) step, followedby culturing on selection medium containing auxin(s) allowingdedifferentiation of cells to form callus. During this callusing phase,transformed resistant callus tissue is selected in the presence of anappropriate selection agent on a selection medium. This is followed bygrowth of cells under conditions that promote differentiation of thecallus and regeneration of the callus into plants on regeneration androoting media. This process has typically required at least 10-12 weeksto produce plants that can be transferred to soil for further growth.The process also requires several manual transfers of tissue throughoutthe transformation process and uses several different types of media.

Thus the use of standard transformation and regeneration protocols istime consuming and inefficient, and negatively impacts transgenicproduct development timelines, given that there is usually a seasonallylimited “priority development window” for making decisions regardingwhich genetic constructs to prioritize for use in larger scale fieldwork based on results obtained during initial research. The availablestandard methods of transformation and regeneration have multipledrawbacks that limit the speed and efficiency with which transgenicplants can be produced and screened. For example, many standard methodsof transformation and regeneration require the use of high auxin orcytokinin levels and require steps involving either embryogenic callusformation or organogenesis, leading to procedures that take many weeksbefore producing plants for growth in a greenhouse setting followingtransformation. It has been reported (Zhong et al. (1992) Planta187:490-497) that such methods can take 12-23 weeks to produce plants,which include the steps of supplying 2,4-D to stimulate somatic embryoformation in corn (taking up to 8 weeks), production of embryogeniccallus from the primary somatic embryos (taking up to an additional 8weeks), forming shoots (taking up to an additional 3 weeks), and finallyrooting (taking up to an additional 1 to 3 weeks). Alternatively, Zhonget al. immediately supplies a cytokinin along with the auxin tostimulate direct morphogenesis to produce shoots and direct plantformation from 8 to 28 weeks (Zhong et al. (1992) Planta 187:490-497).

Despite advances in plant molecular biology, particularly planttransformation and regeneration methods there remains a need for a highthroughput system to produce transgenic plants quickly and efficientlyto provide more time and flexibility for making research and productdevelopment decisions. Such a high throughput system for transformationfacilitates production of large numbers of transgenic plants for genetesting and/or product development while lowering material and laborcosts.

SUMMARY

The present disclosure comprises methods and compositions for the rapidand efficient transformation of plants, e.g., monocot plants such asmaize. In various aspects, the present disclosure further providesmethods for producing a transgenic plant, comprising: (a) transforming acell of an explant with an expression construct comprising (i) anucleotide sequence encoding a WUS/WOX homeobox polypeptide; or (ii) anucleotide sequence encoding a polypeptide comprising two AP2-DNAbinding domains; or (iii) a combination of (i) and (ii); and (b)allowing expression of the polypeptide of (a) in each transformed cellto form a regenerable plant structure in the absence of exogenouscytokinin, wherein no callus is formed; and (c) germinating theregenerable plant structure to form the transgenic plant. In an aspect,the regenerable plant structure is produced within about 0 to about 7days or within about 0 to about 14 days of transforming the cell. In anaspect, germinating comprises transferring the regenerable plantstructure to a maturation medium comprising an exogenous cytokinin andforming the transgenic plant. In an aspect, the expression constructfurther comprises a nucleotide sequence encoding a site-specificrecombinase selected from FLP, Cre, SSV1, lambda Int, phi C31 Int,HK022, R. Gin, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, or U153. In anaspect, the nucleotide sequence encoding a site-specific recombinase isoperably linked to a constitutive promoter, an inducible promoter, or adevelopmentally regulated promoter. In an aspect, (c) germinating isperformed in the presence of exogenous cytokinin. In an aspect, thenucleotide sequence encoding the WUS/WOX homeobox polypeptide and/or thenucleotide sequence encoding a polypeptide comprising two AP2-DNAbinding domains is operably linked to a promoter selected from aninducible promoter, a developmentally regulated promoter, or aconstitutive promoter. In an aspect, the constitutive promoter operablylinked to the nucleotide sequence encoding the WUS/WOX homeoboxpolypeptide and/or the nucleotide sequence encoding a polypeptidecomprising two AP2-DNA binding domains is selected from UBI, LLDAV,EVCV, DMMV, BSV(AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO(ALT1), USB1ZM PRO. ZM-GOS2 PRO, ZM-H1B PRO (1.2 KB), IN2-2, NOS, the-135 version of 35S, or ZM-ADF PRO (ALT2); the inducible promoteroperably linked to the nucleotide sequence encoding the WUS/WOX homeoboxpolypeptide and/or the nucleotide sequence encoding a polypeptidecomprising two AP2-DNA binding domains is selected from AXIG1, DR5, XVE,GLB1, OLE, LTP2, HSP17.7, HSP26, HSP18A, or promoters activated bytetracycline, ethamethsulfuron or chlorsulfuron; and the developmentallyregulated promoter operably linked to the nucleotide sequence encodingthe WUS/WOX homeobox polypeptide and/or the nucleotide sequence encodinga polypeptide comprising two AP2-DNA binding domains is selected fromPLTP, PLTP1, PLTP2, PLTP3, SDR, LGL, LEA-14A, or LEA-D34. In an aspect,the explant is derived from a monocot or a dicot. In an aspect, a seedfrom the plant produced by the method.

In various aspects, the present disclosure further provides methods forproducing a transgenic plant, comprising: (a) transforming a cell of anexplant with an expression construct comprising (i) a nucleotidesequence encoding a WUS/WOX homeobox polypeptide; or (ii) a nucleotidesequence encoding a polypeptide comprising two AP2-DNA binding domains;or (iii) a combination of (i) and (ii); and (b) allowing expression of apolypeptide of (a) in each transformed cell to form a regenerable plantstructure in the absence of exogenous cytokinin, wherein no callus isformed; and; (c) germinating the regenerable plant structure to form thetransgenic plant; wherein the WUS/WOX homeobox polypeptide comprises theamino acid sequence of any of SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16; orwherein the WUS/WOX homeobox polypeptide is encoded by the nucleotidesequence of any of SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15; and wherein thepolypeptide comprising two AP2-DNA binding domains comprises the aminoacid sequence of any of SEQ ID NO: 18, 20, 63, 65, or 67; or wherein thepolypeptide comprising two AP2-DNA binding domains is encoded by thenucleotide sequence of any of SEQ ID NO; 17, 19, 21, 62, 64, 66, or 68.In an aspect, the regenerable plant structure is produced within about 0to about 7 days or within about 0 to about 14 days of transforming thecell. In an aspect, germinating comprises transferring the regenerableplant structure to a maturation medium comprising an exogenous cytokininand forming the transgenic plant. In an aspect, the expression constructfurther comprises a nucleotide sequence encoding a site-specificrecombinase selected from FLP, Cre, SSV1, lambda Int, phi C31 Int,HK022, R. Gin, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, or U153. In anaspect, the nucleotide sequence encoding a site-specific recombinase isoperably linked to a constitutive promoter, an inducible promoter, or adevelopmentally regulated promoter. In an aspect, (c) germinating isperformed in the presence of exogenous cytokinin. In an aspect, thenucleotide sequence encoding the WUS/WOX homeobox polypeptide and/or thenucleotide sequence encoding a polypeptide comprising two AP2-DNAbinding domains is operably linked to a promoter selected from aninducible promoter, a developmentally regulated promoter, or aconstitutive promoter. In an aspect, the constitutive promoter operablylinked to the nucleotide sequence encoding the WUS/WOX homeoboxpolypeptide and/or the nucleotide sequence encoding a polypeptidecomprising two AP2-DNA binding domains is selected from UBI, LLDAV,EVCV, DMMV, BSV(AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO(ALT1), USB1ZM PRO, ZM-GOS2 PRO, ZM-HIB PRO (1.2 KB), IN2-2, NOS, the-135 version of 35S, or ZM-ADF PRO (ALT2); the inducible promoteroperably linked to the nucleotide sequence encoding the WUS/WOX homeoboxpolypeptide and/or the nucleotide sequence encoding a polypeptidecomprising two AP2-DNA binding domains is selected from AXIG1, DR5, XVE,GLB1, OLE, LTP2, HSP17.7, HSP26, HSP18A, or promoters activated bytetracycline, ethamethsulfuron or chlorsulfuron; and the developmentallyregulated promoter operably linked to the nucleotide sequence encodingthe WUS/WOX homeobox polypeptide and/or the nucleotide sequence encodinga polypeptide comprising two AP2-DNA binding domains is selected fromPLTP, PLTP1, PLTP2, PLTP3, SDR, LGL, LEA-14A, or LEA-D34. In an aspect,the explant is derived from a monocot or a dicot. In an aspect, a seedfrom the plant produced by the method.

In various aspects, the present disclosure further provides methods forproducing a transgenic plant, comprising: (a) transforming a cell of anexplant with an expression construct comprising (i) a nucleotidesequence encoding a WUS/WOX homeobox polypeptide; or (ii) a nucleotidesequence encoding a polypeptide comprising two AP2-DNA binding domains;or (iii) a combination of (i) and (ii); (b) allowing expression of thepolypeptide of (a) in each transformed cell to form a regenerable plantstructure in the absence of exogenous cytokinin, wherein no callus isformed; and (c) germinating the regenerable plant structure of (b) forabout 14 to about 60 days to form a plantlet; and (d) allowing theplantlet of (c) to grow into a plant. In an aspect, the regenerableplant structure is produced within about 0 to about 7 days or withinabout 0 to about 14 days of transforming the cell. In an aspect,germinating comprises transferring the regenerable plant structure to amaturation medium comprising an exogenous cytokinin and forming thetransgenic plant. In an aspect, the expression construct furthercomprises a nucleotide sequence encoding a site-specific recombinaseselected from FLP, Cre, SSV1, lambda Int, phi C31 Int, HK022, R, Gin,Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, or U153. In an aspect, thenucleotide sequence encoding a site-specific recombinase is operablylinked to a constitutive promoter, an inducible promoter, or adevelopmentally regulated promoter. In an aspect, (c) germinating isperformed in the presence of exogenous cytokinin. In an aspect, thenucleotide sequence encoding the WUS/WOX homeobox polypeptide and/or thenucleotide sequence encoding a polypeptide comprising two AP2-DNAbinding domains is operably linked to a promoter selected from aninducible promoter, a developmentally regulated promoter, or aconstitutive promoter. In an aspect, the constitutive promoter operablylinked to the nucleotide sequence encoding the WUS/WOX homeoboxpolypeptide and/or the nucleotide sequence encoding a polypeptidecomprising two AP2-DNA binding domains is selected from UBI, LLDAV,EVCV, DMMV, BSV(AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO(ALT1), USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO (1.2 KB), IN2-2, NOS, the-135 version of 35S, or ZM-ADF PRO (ALT2); the inducible promoteroperably linked to the nucleotide sequence encoding the WUS/WOX homeoboxpolypeptide and/or the nucleotide sequence encoding a polypeptidecomprising two AP2-DNA binding domains is selected from AXIG1, DR5, XVE,GLB1, OLE, LTP2, HSP17.7, HSP26, HSP18A, or promoters activated bytetracycline, ethamethsulfuron or chlorsulfuron; and the developmentallyregulated promoter operably linked to the nucleotide sequence encodingthe WUS/WOX homeobox polypeptide and/or the nucleotide sequence encodinga polypeptide comprising two AP2-DNA binding domains is selected fromPLTP, PLTP1, PLTP2, PLTP3, SDR, LGL, LEA-14A, or LEA-D34. In an aspect,the explant is derived from a monocot or a dicot. In an aspect, a seedfrom the plant produced by the method.

In various aspects, the present disclosure further provides methods forproducing a transgenic plant, comprising (a) transforming one or morecells of an explant with an expression construct comprising (i) anucleotide sequence encoding a WUS/WOX homeobox polypeptide; or (ii) anucleotide sequence encoding a polypeptide comprising two AP2-DNAbinding domains; or (iii) a combination of (i) and (ii); and (b)allowing expression of the polypeptide of (a) in each transformed cellto form a regenerable plant structure in the absence of exogenouscytokinin, wherein no callus is formed, and germinating the regenerableplant structure to form the transgenic plant. In an aspect, the presentdisclosure further provides methods for producing a plantlet obtainedfrom a regenerable plant structure prepared using the disclosed methods.In an aspect, the present disclosure further provides methods forproducing a plant obtained from the regenerable plant structure. Invarious aspects, germinating is performed in the presence of exogenouscytokinin. In an aspect, the present disclosure further provides kitscomprising (a) an expression construct comprising (i) a nucleotidesequence encoding a WUS/WOX homeobox polypeptide; or (ii) a nucleotidesequence encoding a polypeptide comprising two AP2-DNA binding domains;or (iii) a combination of (i) and (ii); and (b) instructions forobtaining a plant regenerable structure in the absence of exogenouscytokinin, wherein no callus is formed; and (c) instructions forgerminating the regenerable plant structure to form a transgenic plant.

The present disclosure comprises methods and compositions for the rapidand efficient transformation of plants, e.g., monocot plants such asmaize. In various aspects, the present disclosure provides methods forproducing a transgenic plant, comprising (a) transforming a cell of anexplant with an expression construct comprising (i) a nucleotidesequence encoding a WUS/WOX homeobox polypeptide; or (ii) a nucleotidesequence encoding a polypeptide comprising two AP2-DNA binding domains;or (iii) a combination of (i) and (ii); and (b) allowing expression ofthe polypeptide of (a) in each transformed cell to form a regenerableplant structure in the absence of exogenous cytokinin, wherein no callusis formed; and (c) germinating the regenerable plant structure to formthe transgenic plant. In various aspects, the regenerable plantstructure is produced within about 0-7 days or about 0-14 days oftransforming the cell. In various aspects, germinating comprisestransferring the regenerable plant structure to a maturation mediumcomprising an exogenous cytokinin and forming the transgenic plant. Invarious aspects, germinating is performed in the presence of exogenouscytokinin. In various aspects, the expression construct furthercomprises a nucleotide sequence encoding a site-specific recombinaseselected from of FLP, Cre, SSV1, lambda Int, phi C31 Int, HK022, R, Gin,Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, or U153. In various aspects,the nucleotide sequence encoding a site-specific recombinase is operablylinked to a constitutive promoter, an inducible promoter, or adevelopmentally-regulated promoter. In various aspects, the induciblepromoter is GLB1, OLE, LTP2, HSP17.7, HSP26, HSP18A or XVE. In variousaspects, the nucleotide sequence encoding the WUS/WOX homeoboxpolypeptide is operably linked to a promoter selected from an induciblepromoter, a developmentally regulated promoter, or a constitutivepromoter. In various aspects, the constitutive promoter is selected fromUBI, LLDAV, EVCV, DMMV, BSV (AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3PRO, SB-UBI PRO (ALT1), USB1ZM PRO, ZM-GOS2 PRO, ZM-HIB PRO (1.2 KB),IN2-2, NOS, the -135 version of 35S, or ZM-ADF PRO (ALT2); the induciblepromoter is selected from GLB1, OLE, LTP2, AXIG1 DR5, HSP17.7, HSP26,HSP18A, or XVE; and the developmentally regulated promoter is selectedfrom PLTP, PLTP1, PLTP2, PLTP3, LGL, LEA-14A, or LEA-D34. In variousaspects, the explant is derived from a monocot or a dicot. In variousaspects, a seed from the plant produced by the method is provided.

In a further aspect, the present disclosure provides methods forproducing a transgenic plant, comprising (a)transforming a cell of anexplant with an expression construct comprising (i) a nucleotidesequence encoding a WUS/WOX homeobox polypeptide; or (ii) a nucleotidesequence encoding a polypeptide comprising two AP2-DNA binding domains;or (iii) a combination thereof; and (b) allowing expression of apolypeptide of (a) in each transformed cell to form a regenerable plantstructure in the absence of exogenous cytokinin, wherein no callus isformed; and; (c)

germinating the regenerable plant structure to form the transgenicplant; wherein the WUS/WOX homeobox polypeptide comprises the amino acidsequence of any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16; or whereinthe WUS/WOX homeobox polypeptide is encoded by the nucleotide sequenceof any one of SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15; and wherein thepolypeptide comprising two AP2-DNA binding domains comprises the aminoacid sequence of any one of SEQ ID NO; 18, 20, 63, 65, or 67; or whereinthe polypeptide comprising two AP2-DNA binding domains is encoded by thenucleotide sequence of any one of SEQ ID NO: 17, 19, 21, 62, 64, 66, or68. In various aspects, the regenerable plant structure is producedwithin about 0-7 days or about 0-14 days of transforming the cell. Invarious aspects, germinating comprises transferring the regenerableplant structure to a maturation medium comprising an exogenous cytokininand forming the transgenic plant. In various aspects, germinating isperformed in the presence of exogenous cytokinin. In various aspects,the expression construct further comprises a nucleotide sequenceencoding a site-specific recombinase selected from FLP, Cre, SSV1,lambda Int, phi C31 Int, HK022, R, Gin, Tn1721, CinH, ParA, Tn5053,Bxb1, TP907-1, or U153. In various aspects, the nucleotide sequenceencoding a site-specific recombinase is operably linked to aconstitutive promoter, an inducible promoter, or a developmentallyregulated promoter. In various aspects, the inducible promoter is GLB1,OLE, LTP2, HSP17.7, HSP26, HSP18A or XVE. In various aspects, thenucleotide sequence encoding the WUS/WOX homeobox polypeptide isoperably linked to a promoter selected from an inducible promoter, adevelopmentally regulated promoter, or a constitutive promoter. Invarious aspects, the constitutive promoter is selected from UBI, LLDAV,EVCV, DMMV, BSV (AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO(ALT1), USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO (1.2 KB), IN2-2, NOS, the-135 version of 35S, or ZM-ADF PRO (ALT2); the inducible promoter isselected from GLB1, OLE, LTP2, AXIG1 DR5, HSP17.7, HSP26, HSP18A or XVE;and the developmentally regulated promoter is selected from PLTP, PLTP1,PLTP2, PLTP3, LGL, LEA-14A, or LEA-D34. In various aspects, the explantis derived from a monocot or a dicot. In various aspects, a seed fromthe plant produced by the method is provided.

In a further aspect, the present disclosure provides methods forproducing a transgenic plant comprising: (a) transforming a cell of anexplant with an expression construct comprising (i) a nucleotidesequence encoding a WUS/WOX homeobox polypeptide; (ii) a nucleotidesequence encoding a polypeptide comprising two AP2-DNA binding domains;or (iii) a combination of (i) and (ii); (b) allowing expression of thepolypeptide of (a) in each transformed cell to form a regenerable plantstructure in the absence of exogenous cytokinin, wherein no callus isformed; and (c) allowing the regenerable plant structure of (b) tomature into a plantlet for about 14 to about 60 days; and (d) allowingthe plantlet of (c) to grow into a plant. In various aspects, theregenerable plant structure is produced within about 0-7 days or about0-14 days of transforming the cell. In various aspects, germinatingcomprises transferring the regenerable plant structure to a maturationmedium comprising an exogenous cytokinin and forming the transgenicplant. In various aspects, germinating is performed in the presence ofexogenous cytokinin. In various aspects, the expression constructfurther comprises a nucleotide sequence encoding a site-specificrecombinase selected from FLP, Cre, SSV1, lambda Int, phi C31 Int,HK022, R, Gin, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, or U153. Invarious aspects, the nucleotide sequence encoding a site-specificrecombinase is operably linked to a constitutive promoter, an induciblepromoter, or a developmentally-regulated promoter. In various aspects,the inducible promoter is GLB1, OLE, LTP2, HSP17.7. HSP26, HSP18A orXVE. In various aspects, the nucleotide sequence encoding the WUS/WOXhomeobox polypeptide is operably linked to a promoter selected from aninducible promoter, a developmentally regulated promoter, and aconstitutive promoter. In various aspects, the constitutive promoter isselected from UBI, LLDAV, EVCV, DMMV, BSV (AY) PRO, CYMV PRO FL, UBIZMPRO, SI-UB3 PRO, SB-UBI PRO (ALT1), USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO(1.2 KB), IN2-2, NOS, the -135 version of 35S, and ZM-ADF PRO (ALT2);the inducible promoter is selected from GLB1, OLE, LTP2, AXIG1 DR5,HSP17.7, HSP26, HSP18A, or XVE; and the developmentally regulatedpromoter is selected from PLTP, PLTP1, PLTP2, PLTP3, LGL, LEA-14A, orLEA-D34. In various aspects, the explant is derived from a monocot or adicot. In various aspects, a seed from the plant produced by the methodis provided.

In a further aspect, the present disclosure provides methods forproducing a transgenic plant, comprising (a) transforming a cell of anexplant with an expression construct comprising (i) a nucleotidesequence encoding a WUS/WOX homeobox polypeptide; or (ii) a nucleotidesequence encoding a polypeptide comprising two AP2-DNA binding domains;or (iii) a combination of (i) and (ii); and (b) allowing expression ofthe polypeptide of (a) in each transformed cell to form a regenerableplant structure in the absence of exogenous cytokinin, wherein no callusis formed; and (c) germinating the regenerable plant structure to formthe transgenic plant. In various aspects, the expression constructcomprises both the nucleotide sequence encoding the WUS/WOX homeoboxpolypeptide and the nucleotide sequence encoding the polypeptidecomprising two AP2-DNA binding domains. In various aspects, theexpression construct comprises the nucleotide sequence encoding theWUS/WOX homeobox polypeptide. In various aspects, the expressionconstruct comprises the nucleotide sequence encoding the polypeptidecomprising two AP2-DNA binding domains. In various aspects, theexpression of the nucleotide sequence encoding the WUS/WOX homeoboxpolypeptide and/or the nucleotide sequence encoding the polypeptidecomprising two AP2 binding domains occurs less than 1 day, less than 2days, less than 5 days, less than 7 days, or less than about 14 daysafter initiation of transformation. In various aspects, germinating isperformed in the presence of exogenous cytokinin. In various aspects,the expression construct further comprises a nucleotide sequenceencoding a site-specific recombinase. In various aspects, thesite-specific recombinase is FLP, Cre, SSV1, lambda Int, phi C31 Int,HK022, R, Gin. Tn1721, CinH, ParA, Tn5053. Bxb1, TP907-1, or U153. Invarious aspects, the site-specific recombinase is a destabilized fusionpolypeptide. In various aspects, the destabilized fusion polypeptide isTETR(L17G)˜CRE or ESR(L17G)˜CRE. In various aspects, the nucleotidesequence encoding a site-specific recombinase is operably linked to aconstitutive promoter, an inducible promoter, or adevelopmentally-regulated promoter. In various aspects, the induciblepromoter is GLB1, OLE, LTP2, HSP17.7, HSP26, or HSP18A. In variousaspects, the inducible promoter is a chemically-inducible promoter. Invarious aspects, the chemically-inducible promoter is XVE. In variousaspects, the chemically-inducible promoter is repressed by TETR, ESR, orCR, and de-repression occurs upon addition of tetracycline-related orsulfonylurea ligands. In various aspects, the repressor is TETR and thetetracycline-related ligand is doxycycline or anhydrotetracycline. Invarious aspects, the repressor is ESR and the sulfonylurea ligand isethametsulfuron, chlorsulfuron, metsulfuron-methyl, sulfometuron methyl,chlorimuron ethyl, nicosulfuron, primisulfuron, tribenuron,sulfosulfuron, trifloxysulfuron, foramsulfuron, iodosulfuron,prosulfuron, thifensulfuron, rimsulfuron, mesosulfuron, or halosulfuron.In various aspects, the nucleotide sequence encoding the WUS/WOXhomeobox polypeptide is operably linked to a constitutive promoter. Invarious aspects, the constitutive promoter is UBI, LLDAV, EVCV, DMMV,BSV (AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO (ALT1),USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO (1.2 KB), IN2-2, NOS, the -135version of 35S, or ZM-ADF PRO (ALT2). In various aspects, the nucleotidesequence encoding the WUS/WOX homeobox polypeptide is operably linked toan inducible promoter or a developmentally-regulated promoter. Invarious aspects, the nucleotide sequence encoding a polypeptidecomprising two AP2-DNA binding domains is operably linked to aninducible promoter or a developmentally-regulated promoter. In variousaspects, the developmentally-regulated promoter is a PLTP promoter. Invarious aspects, the PLTP promoter is derived from a monocot. In variousaspects, the monocot is barley, maize, millet, oats, rice, rye, Setariasp., sorghum, sugarcane, switchgrass, triticale, turfgrass, or wheat. Invarious aspects, the monocot is maize, sorghum, rice, or Setaria sp. Invarious aspects, the monocot is maize, sorghum or rice. In variousaspects, the monocot is maize. In various aspects, the PLTP promoter isderived from a dicot. In various aspects, the dicot is kale,cauliflower, broccoli, mustard plant, cabbage, pea, clover, alfalfa,broad bean, tomato, cassava, soybean, canola, alfalfa, sunflower,safflower, tobacco. Arabidopsis, or cotton. In various aspects, the PLTPpromoter comprises any one of SEQ ID NO: 1-2 or 55-61. In variousaspects, the PLTP promoter comprises any one of SEQ ID NO: 1 or 2. Invarious aspects, the inducible promoter is an auxin-inducible promoter.In various aspects, the auxin inducible promoter is an AXIG1. In variousaspects, the AXIG1 promoter comprises the nucleotide sequence of SEQ IDNO: 39. In various aspects, the promoter comprises an auxin-responseelement. In various aspects, the promoter contains one or more DR5enhancer motifs. In various aspects, the promoter is a weak constitutivepromoter modified for repression and de-repression. In various aspects,one or more operator sequences in the promoter have been positioned nearor overlapping the TATA box and/or the transcription start site. Invarious aspects, the promoter is NOS. AXIG1, ZM-GOS2. CC-UB11-PRO orZM-ADF4-PRO. In various aspects, the promoter is a DR5 promotercomprising the nucleotide sequence of SEQ ID NO: 40. In various aspects,the promoter is a de-repressible promoter. In various aspects, thede-repressible promoter is TETR, ESR, or CR. In various aspects, theWUS/WOX homeobox polypeptide is a WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5,or WOX9 polypeptide. In various aspects, the nucleotide sequenceencoding the WUS/WOX homeobox polypeptide is a monocot nucleotide. Invarious aspects, the monocot is barley, maize, millet, oats, rice, rye,Setaria sp., sorghum, sugarcane, switchgrass, triticale, turfgrass, orwheat. In various aspects, the nucleotide sequence encoding the WUS/WOXhomeobox polypeptide is a dicot nucleotide. In various aspects, thedicot is kale, cauliflower, broccoli, mustard plant, cabbage, pea,clover, alfalfa, broad bean, tomato, cassava, soybean, canola, alfalfa,sunflower, safflower, tobacco, Arabidopsis, or cotton. In variousaspects, the nucleotide sequence encoding a WUS/WOX homeobox polypeptideencodes an amino acid sequence comprising any one of SEQ ID NOs:4, 6, 8,10, 12, 14, or 16. In various aspects, the nucleotide sequence encodinga WUS/WOX homeobox polypeptide comprises any one of SEQ ID NOs:3, 5, 7,9, 11, 13, or 15. In various aspects, the WUS/WOX homeobox polypeptideis a WUS1 polypeptide. In various aspects, the WUS1 polypeptide is amaize, sorghum, rice or Setaria sp. WUS1 polypeptide. In variousaspects, the WUS1 polypeptide is a maize or rice WUS1 polypeptide. Invarious aspects, the WUS1 polypeptide is a maize WUS1 polypeptide. Invarious aspects, the WUS1 polypeptide comprises an amino acid sequenceof SEQ ID NO: 4. In various aspects, the WUS1 polypeptide is encoded bya nucleotide sequence comprising SEQ ID NO: 3. In various aspects, theWUS/WOX homeobox polypeptide is a WUS2 polypeptide. In various aspects,the WUS2 polypeptide is a maize, sorghum, rice or Setaria sp. WUS2polypeptide. In various aspects, the WUS2 polypeptide is a maize or riceWUS2 polypeptide. In various aspects, the WUS2 polypeptide is a maizeWUS2 polypeptide. In various aspects, the WUS2 polypeptide comprises theamino acid sequence SEQ ID NO:6. In various aspects, the WUS2polypeptide is encoded by a nucleotide sequence comprising SEQ ID NO: 5.In various aspects, the WUS/WOX homeobox polypeptide is a WUS3polypeptide. In various aspects, the WUS3 polypeptide is a maize,sorghum, rice or Setaria WUS3 polypeptide. In various aspects, the WUS3polypeptide is a maize or rice WUS3 polypeptide. In various aspects, theWUS3 polypeptide is a maize WUS3 polypeptide. In various aspects, theWUS3 polypeptide comprises the amino sequence of SEQ ID NO:8. In variousaspects, the WUS3 polypeptide is encoded by a nucleotide sequencecomprising SEQ ID NO:7. In various aspects, the WUS/WOX homeoboxpolypeptide is a WOX5 polypeptide. In various aspects, the WOX5polypeptide is a WOX5A polypeptide. In various aspects, the WOX5polypeptide is a maize, sorghum, rice or Setaria WOX5 polypeptide. Invarious aspects, the WOX5 polypeptide is a maize or rice WOX5polypeptide. In various aspects, the WOX5 polypeptide is a maize WOX5polypeptide. In various aspects, the WOX5 polypeptide comprises theamino acid sequence of SEQ ID NO: 14. In various aspects, the WOX5polypeptide is encoded by a nucleotide sequence comprising SEQ ID NO:13. In various aspects, the polypeptide comprising the two AP2-DNAbinding domains is an ODP2, BBM2, BMN2, or BMN3 polypeptide. In variousaspects, the polypeptide comprising the two AP2-DNA binding domains is amonocot polypeptide. In various aspects, the monocot is barley, maize,millet, oats, rice, rye, Setaria sp., sorghum, sugarcane, switchgrass,triticale, turfgrass, or wheat. In various aspects, the monocot ismaize, sorghum, rice, or Setaria sp. In various aspects, the monocot ismaize or rice. In various aspects, the monocot is maize. In variousaspects, the polypeptide comprising the two AP2-DNA binding domains is adicot polypeptide. In various aspects, the dicot is kale, cauliflower,broccoli, mustard plant, cabbage, pea, clover, alfalfa, broad bean,tomato, cassava, soybean, canola, alfalfa, sunflower, safflower,tobacco, Arabidopsis, or cotton. In various aspects, the polypeptidecomprising the two AP2-DNA binding domains comprises an amino acidsequence of any one of SEQ ID NO: 18, 20, 63, 65, or 67. In variousaspects, the polypeptide comprising the two AP2-DNA binding domains isencoded by a nucleotide sequence comprising any one of SEQ ID NO: 17,19, 21, 62, 64, 66, or 68. In various aspects, the polypeptidecomprising the two AP2-DNA binding domains is an ODP2 polypeptide. Invarious aspects, the ODP2 polypeptide is a monocot polypeptide. Invarious aspects, the monocot is barley, maize, millet, oats, rice, rye,Setaria sp., sorghum, sugarcane, switchgrass, triticale, turfgrass, orwheat. In various aspects, the monocot is maize, rice, or Setaria sp. Invarious aspects, the monocot is maize or rice. In various aspects, theODP2 is a dicot polypeptide. In various aspects, the dicot is kale,cauliflower, broccoli, mustard plant, cabbage, pea, clover, alfalfa,broad bean, tomato, cassava, soybean, canola, alfalfa, sunflower,safflower, tobacco, Arabidopsis, or cotton. In various aspects, the ODP2polypeptide comprises the amino acid sequence of any one of SEQ ID NO:18, 63, 65, or 67. In various aspects, the ODP2 polypeptide is encodedby a nucleotide sequence comprising the sequence of any one of SEQ IDNO: 17, 21, 62, 64, 66, or 68. In various aspects, the polypeptidecomprising the two AP2-DNA binding domains is a BBM2 polypeptide. Invarious aspects, the BBM2 polypeptide is a monocot polypeptide. Invarious aspects, the monocot is barley, maize, millet, oats, rice, rye,Setaria sp., sorghum, sugarcane, switchgrass, triticale, turfgrass, orwheat. In various aspects, the monocot is maize, sugarcane, rice, orSetaria sp. In various aspects, the monocot is maize or rice. In variousaspects, the monocot is maize. In various aspects, the BBM2 polypeptideis a dicot polypeptide. In various aspects, the dicot is kale,cauliflower, broccoli, mustard plant, cabbage, pea, clover, alfalfa,broad bean, tomato, cassava, soybean, canola, alfalfa, sunflower,safflower, tobacco, Arabidopsis, or cotton. In various aspects, the BBM2polypeptide comprises the amino acid sequence of SEQ ID NO: 20. Invarious aspects, the BBM2 polypeptide is encoded by a nucleotidesequence comprising the sequence of SEQ ID NO: 19. In various aspects,the explant is derived from a monocot. In various aspects, the monocotis barley, maize, millet, oats, rice, rye, Setaria sp., sorghum,sugarcane, switchgrass, triticale, turfgrass, or wheat. In variousaspects, the explant is derived from a dicot. In various aspects, thedicot is kale, cauliflower, broccoli, mustard plant, cabbage, pea,clover, alfalfa, broad bean, tomato, cassava, soybean, canola, alfalfa,sunflower, safflower, tobacco, Arabidopsis, or cotton. In variousaspects, the regenerable plant structure is formed within about 0 toabout 7 days of transforming the cell or within about 0 to about 14 daysof transforming the cell and the transgenic plant is formed in about 14days of transforming the cell to about 60 days of transforming the cell.In various aspects, the method is carried out in the absence of rootingmedium. In various aspects, the method is carried out in the presence ofrooting medium. In various aspects, the explant is an immature embryo.In various aspects, the immature embryo is a 1-5 mm immature embryo. Invarious aspects, the immature embryo is a 3.5-5 mm immature embryo. Invarious aspects, exogenous cytokinin is used during germinating afterabout 7 days of transforming the cell or after about 14 days oftransforming the cell. In various aspects, the expression of apolypeptide of (a) is transient. In various aspects, a seed from theplant produced by the method is provided.

In a further aspect, the present disclosure provides methods forproducing a transgenic plant, comprising (a) transforming a cell of anexplant with an expression construct comprising (i) a nucleotidesequence encoding a WUS/WOX homeobox polypeptide; or (ii) a nucleotidesequence encoding a polypeptide comprising two AP2-DNA binding domains;or (iii) a combination thereof; (b) allowing expression of a polypeptideof (a) in each transformed cell to form a regenerable plant structure inthe absence of exogenous cytokinin, wherein no callus is formed; and (c)germinating the regenerable plant structure to form the transgenicplant; wherein the WUS/WOX homeobox polypeptide comprises the amino acidsequence of any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16; or whereinthe WUS/WOX homeobox polypeptide is encoded by the nucleotide sequenceof any one of SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15; and wherein thepolypeptide comprising two AP2-DNA binding domains comprises the aminoacid sequence of any one of SEQ ID NO: 18, 20, 63, 65, or 67; or whereinthe polypeptide comprising two AP2-DNA binding domains is encoded by thenucleotide sequence of any one of SEQ ID NO: 17, 19, 21, 62, 64, 66, or68. In various aspects, the expression construct comprises both thenucleotide sequence encoding the WUS/WOX homeobox polypeptide and thenucleotide sequence encoding the polypeptide comprising two AP2-DNAbinding domains. In various aspects, the expression construct comprisesthe nucleotide sequence encoding the WUS/WOX homeobox polypeptide. Invarious aspects, the expression construct comprises the nucleotidesequence encoding the polypeptide comprising two AP2-DNA bindingdomains. In various aspects, the expression of the nucleotide sequenceencoding the WUS/WOX homeobox polypeptide and/or the nucleotide sequenceencoding the polypeptide comprising two AP2 binding domains occurs lessthan 1 day, less than 2 days, less than 5 days, less than 7 days, orless than about 14 days after initiation of transformation. In variousaspects, the expression construct further comprises a nucleotidesequence encoding a site-specific recombinase. In various aspects, thesite-specific recombinase is FLP, Cre, SSV1, lambda Int, phi C31 Int,HK022, R, Gin, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, or U153. Invarious aspects, the site-specific recombinase is a destabilized fusionpolypeptide. In various aspects, the destabilized fusion polypeptide isTETR(L17G)˜CRE or ESR(L17G)˜CRE. In various aspects, the nucleotidesequence encoding a site-specific recombinase is operably linked to aconstitutive promoter, an inducible promoter, or adevelopmentally-regulated promoter. In various aspects, the induciblepromoter is GLB1, OLE, LTP2, HSP17.7, HSP26, or HSP18A. In variousaspects, the inducible promoter is a chemically-inducible promoter. Invarious aspects, the chemically-inducible promoter is XVE. In variousaspects, the chemically-inducible promoter is repressed by TETR, ESR, orCR, and de-repression occurs upon addition of tetracycline-related orsulfonylurea ligands. In various aspects, the repressor is TETR and thetetracycline-related ligand is doxycycline or anhydrotetracycline. Invarious aspects, the repressor is ESR and the sulfonylurea ligand isethametsulfuron, chlorsulfuron, metsulfuron-methyl, sulfometuron methyl,chlorimuron ethyl, nicosulfuron, primisulfuron, tribenuron,sulfosulfuron, trifloxysulfuron, foramsulfuron, iodosulfuron,prosulfuron, thifensulfuron, rimsulfuron, mesosulfuron, or halosulfuron.In various aspects, the nucleotide sequence encoding the WUS/WOXhomeobox polypeptide is operably linked to a constitutive promoter. Invarious aspects, the constitutive promoter is UBI, LLDAV, EVCV, DMMV,BSV (AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO (ALT1),USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO (1.2 KB), IN2-2, NOS, the -135version of 35S, or ZM-ADF PRO (ALT2). In various aspects, the nucleotidesequence encoding the WUS/WOX homeobox polypeptide is operably linked toan inducible promoter or a developmentally-regulated promoter. Invarious aspects, the nucleotide sequence encoding a polypeptidecomprising two AP2-DNA binding domains is operably linked to aninducible promoter or a developmentally-regulated promoter. In variousaspects, the developmentally-regulated promoter is a PLTP promoter. Invarious aspects, the PLTP promoter is derived from a monocot. In variousaspects, the monocot is barley, maize, millet, oats, rice, rye, Setariasp., sorghum, sugarcane, switchgrass, triticale, turfgrass, or wheat. Invarious aspects, the monocot is maize, sorghum, rice, or Setaria sp. Invarious aspects, the monocot is maize, sorghum or rice. In variousaspects, the monocot is maize. In various aspects, the PLTP promoter isderived from a dicot. In various aspects, the dicot is kale,cauliflower, broccoli, mustard plant, cabbage, pea, clover, alfalfa,broad bean, tomato, cassava, soybean, canola, alfalfa, sunflower,safflower, tobacco, Arabidopsis, or cotton. In various aspects, the PLTPpromoter comprises any one of SEQ ID NO: 1-2 or 55-61. In variousaspects, the PLTP promoter comprises any one of SEQ ID NO: 1 or 2. Invarious aspects, the inducible promoter is an auxin-inducible promoter.In various aspects, the auxin inducible promoter is an AXIG1. In variousaspects, the AXIG1 promoter comprises the nucleotide sequence of SEQ IDNO: 39. In various aspects, the promoter comprises an auxin-responseelement. In various aspects, the promoter contains one or more DR5enhancer motifs. In various aspects, the promoter is a weak constitutivepromoter modified for repression and de-repression. In various aspects,one or more operator sequences in the promoter have been positioned nearor overlapping the TATA box and/or the transcription start site. Invarious aspects, the promoter is NOS, AXIG1. ZM-GOS2, CC-UBI1-PRO orZM-ADF4-PRO. In various aspects, the promoter is a DR5 promotercomprising the nucleotide sequence of SEQ ID NO: 40. In various aspects,the promoter is a de-repressible promoter. In various aspects, thede-repressible promoter is TETR, ESR, or CR. In various aspects, theWUS/WOX homeobox polypeptide is SEQ ID NO: 4. In various aspects, theWUS/WOX homeobox polypeptide is encoded by SEQ ID NO: 3. In variousaspects, the WUS/WOX homeobox polypeptide is SEQ ID NO:6 In variousaspects, the WUS/WOX homeobox polypeptide is encoded by SEQ ID NO: 5. Invarious aspects, the WUS/WOX homeobox polypeptide is SEQ ID NO:8. Invarious aspects, the WUS/WOX homeobox polypeptide is encoded by SEQ IDNO: 7. In various aspects, the WUS/WOX homeobox polypeptide is SEQ IDNO: 14. In various aspects, the WUS/WOX homeobox polypeptide is encodedby SEQ ID NO: 13. In various aspects, the polypeptide comprising twoAP2-DNA binding domains is SEQ ID NO: 20. In various aspects, thepolypeptide comprising two AP2-DNA binding domains is encoded by SEQ IDNO: 19. In various aspects, the explant is derived from a monocot. Invarious aspects, the monocot is barley, maize, millet, oats, rice, rye,Setaria sp., sorghum, sugarcane, switchgrass, triticale, turfgrass, orwheat. In various aspects, the explant is derived from a dicot. Invarious aspects, the dicot is kale, cauliflower, broccoli, mustardplant, cabbage, pea, clover, alfalfa, broad bean, tomato, cassava,soybean, canola, alfalfa, sunflower, safflower, tobacco, Arabidopsis, orcotton. In various aspects, the regenerable plant structure is formedwithin about 0 to about 7 days of transforming the cell or within about0 to about 14 days of transforming the cell and the transgenic plant isformed in about 14 days of transforming the cell to about 60 days oftransforming the cell. In various aspects, the method is carried out inthe absence of rooting medium. In various aspects, the method is carriedout in the presence of rooting medium. In various aspects, the explantis an immature embryo. In various aspects, the immature embryo is a 1-5mm immature embryo. In various aspects, the immature embryo is a 3.5-5mm immature embryo. In various aspects, exogenous cytokinin is usedduring germinating after about 7 days of transforming the cell or afterabout 14 days of transforming the cell. In various aspects, theexpression of a polypeptide of (a) is transient. In various aspects, aseed from the plant produced by the method is provided.

In a further aspect, the present disclosure provides methods forproducing a transgenic plant, comprising (a)transforming a cell of anexplant with an expression construct comprising (i) a nucleotidesequence encoding a WUS2 polypeptide; or (ii) a nucleotide sequenceencoding an ODP2 polypeptide; or (iii) a combination thereof; (b)allowing expression of a polypeptide of (a) in each transformed cell toform a regenerable plant structure in the absence of exogenouscytokinin, wherein no callus is formed; and (c) germinating theregenerable plant structure to form the transgenic plant; wherein theWUS2 polypeptide comprises the amino acid sequence of SEQ ID NO: 4; orwherein the WUS2 polypeptide is encoded by the nucleotide sequence ofSEQ ID NO: 3; and wherein the ODP2 polypeptide comprises the amino acidsequence of SEQ ID NO: 18; or wherein the polypeptide comprising twoAP2-DNA binding domains is encoded by the nucleotide sequence of any oneof SEQ ID NO: 17 or 21. In various aspects, the expression constructcomprises both the nucleotide sequence encoding the WUS/WOX homeoboxpolypeptide and the nucleotide sequence encoding the polypeptidecomprising two AP2-DNA binding domains. In various aspects, theexpression construct comprises the nucleotide sequence encoding theWUS/WOX homeobox polypeptide. In various aspects, the expressionconstruct comprises the nucleotide sequence encoding the polypeptidecomprising two AP2-DNA binding domains. In various aspects, theexpression of the nucleotide sequence encoding the WUS/WOX homeoboxpolypeptide and/or the nucleotide sequence encoding the polypeptidecomprising two AP2 binding domains occurs less than 1 day, less than 2days, less than 5 days, less than 7 days, or less than about 14 daysafter initiation of transformation. In various aspects, germinating isperformed in the presence of exogenous cytokinin. In various aspects,the expression construct further comprises a nucleotide sequenceencoding a site-specific recombinase. In various aspects, thesite-specific recombinase is FLP, Cre, SSV1, lambda Int, phi C31 Int,HK022, R, Gin, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, or U153. Invarious aspects, the site-specific recombinase is a destabilized fusionpolypeptide. In various aspects, the destabilized fusion polypeptide isTETR(L17G)˜CRE or ESR(L17G)˜CRE. In various aspects, the nucleotidesequence encoding a site-specific recombinase is operably linked to aconstitutive promoter, an inducible promoter, or adevelopmentally-regulated promoter. In various aspects, the induciblepromoter is GLB1, OLE, LTP2, HSP17.7, HSP26, or HSP18A. In variousaspects, the inducible promoter is a chemically-inducible promoter. Invarious aspects, the chemically-inducible promoter is XVE. In variousaspects, the chemically-inducible promoter is repressed by TETR, ESR, orCR, and de-repression occurs upon addition of tetracycline-related orsulfonylurea ligands. In various aspects, the repressor is TETR and thetetracycline-related ligand is doxycycline or anhydrotetracycline. Invarious aspects, the repressor is ESR and the sulfonylurea ligand isethametsulfuron, chlorsulfuron, metsulfuron-methyl, sulfometuron methyl,chlorimuron ethyl, nicosulfuron, primisulfuron, tribenuron,sulfosulfuron, trifloxysulfuron, foramsulfuron, iodosulfuron,prosulfuron, thifensulfuron, rimsulfuron, mesosulfuron, or halosulfuron.In various aspects, the nucleotide sequence encoding the WUS/WOXhomeobox polypeptide is operably linked to a constitutive promoter. Invarious aspects, the constitutive promoter is UBI, LLDAV, EVCV, DMMV,BSV (AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO (ALT1),USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO (1.2 KB), IN2-2, NOS, the -135version of 35S, or ZM-ADF PRO (ALT2). In various aspects, the nucleotidesequence encoding the WUS/WOX homeobox polypeptide is operably linked toan inducible promoter or a developmentally-regulated promoter. Invarious aspects, the nucleotide sequence encoding a polypeptidecomprising two AP2-DNA binding domains is operably linked to aninducible promoter or a developmentally-regulated promoter. In variousaspects, the developmentally-regulated promoter is a PLTP promoter. Invarious aspects, the PLTP promoter is derived from a monocot. In variousaspects, the monocot is barley, maize, millet, oats, rice, rye, Setariasp., sorghum, sugarcane, switchgrass, triticale, turfgrass, or wheat. Invarious aspects, the monocot is maize, sorghum, rice, or Setaria sp. Invarious aspects, the monocot is maize, sorghum or rice. In variousaspects, the monocot is maize. In various aspects, the PLTP promoter isderived from a dicot. In various aspects, the dicot is kale,cauliflower, broccoli, mustard plant, cabbage, pea, clover, alfalfa,broad bean, tomato, cassava, soybean, canola, alfalfa, sunflower,safflower, tobacco, Arabidopsis, or cotton. In various aspects, the PLTPpromoter comprises any one of SEQ ID NO: 1-2 or 55-61. In variousaspects, the PLTP promoter comprises any one of SEQ ID NO: 1 or 2. Invarious aspects, the inducible promoter is an auxin-inducible promoter.In various aspects, the auxin inducible promoter is an AXIG1. In variousaspects, the AXIG1 promoter comprises the nucleotide sequence of SEQ IDNO: 39. In various aspects, the promoter comprises an auxin-responseelement. In various aspects, the promoter contains one or more DR5enhancer motifs. In various aspects, the promoter is a weak constitutivepromoter modified for repression and de-repression. In various aspects,one or more operator sequences in the promoter have been positioned nearor overlapping the TATA box and/or the transcription start site. Invarious aspects, the promoter is NOS, AXIG1, ZM-GOS2, CC-UB11-PRO orZM-ADF4-PRO. In various aspects, the promoter is a DR5 promotercomprising the nucleotide sequence of SEQ ID NO: 40. In various aspects,the promoter is a de-repressible promoter. In various aspects, thede-repressible promoter is TETR, ESR, or CR. In various aspects, theexplant is derived from a monocot. In various aspects, the monocot isbarley, maize, millet, oats, rice, rye, Setaria sp., sorghum, sugarcane,switchgrass, triticale, turfgrass, or wheat. In various aspects, theexplant is derived from a dicot. In various aspects, the dicot is kale,cauliflower, broccoli, mustard plant, cabbage, pea, clover, alfalfa,broad bean, tomato, cassava, soybean, canola, alfalfa, sunflower,safflower, tobacco, Arabidopsis, or cotton. In various aspects, theregenerable plant structure is formed within about 0 to about 7 days oftransforming the cell or within about 0 to about 14 days of transformingthe cell and the transgenic plant is formed in about 14 days oftransforming the cell to about 60 days of transforming the cell. Invarious aspects, the method is carried out in the absence of rootingmedium. In various aspects, the method is carried out in the presence ofrooting medium. In various aspects, the explant is an immature embryo.In various aspects, the immature embryo is a 1-5 mm immature embryo. Invarious aspects, the immature embryo is a 3.5-5 mm immature embryo. Invarious aspects, exogenous cytokinin is used during germinating afterabout 7 days of transforming the cell or after about 14 days oftransforming the cell. In various aspects, the expression of apolypeptide of (a) is transient. In various aspects, a seed from theplant produced by the method is provided.

In a further aspect, the present disclosure provides methods forproducing a transgenic plant, comprising (a) transforming a cell of anexplant with an expression construct comprising (i) a nucleotidesequence encoding a WUS/WOX homeobox polypeptide; and (b) allowingexpression of the polypeptide of (a) in each transformed cell to form aregenerable plant structure in the absence of exogenous cytokinin,wherein no callus is formed; and (c) germinating the regenerable plantstructure to form the transgenic plant. In various aspects, theexpression of the nucleotide sequence encoding the WUS/WOX homeoboxpolypeptide occurs less than 1 day, less than 2 days, less than 5 days,less than 7 days, or less than about 14 days after initiation oftransformation. In various aspects, germinating is performed in thepresence of exogenous cytokinin. In various aspects, the expressionconstruct further comprises a nucleotide sequence encoding asite-specific recombinase. In various aspects, the site-specificrecombinase is FLP, Cre, SSV1, lambda Int, phi C31 Int, HK022, R, Gin,Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, or U153. In various aspects,the site-specific recombinase is a destabilized fusion polypeptide. Invarious aspects, the destabilized fusion polypeptide is TETR(L17G)˜CREor ESR(L17G)˜CRE. In various aspects, the nucleotide sequence encoding asite-specific recombinase is operably linked to a constitutive promoter,an inducible promoter, or a developmentally-regulated promoter. Invarious aspects, the inducible promoter is GLB1, OLE, LTP2, HSP17.7,HSP26, or HSP18A. In various aspects, the inducible promoter is achemically-inducible promoter. In various aspects, thechemically-inducible promoter is XVE. In various aspects, thechemically-inducible promoter is repressed by TETR, ESR, or CR, andde-repression occurs upon addition of tetracycline-related orsulfonylurea ligands. In various aspects, the repressor is TETR and thetetracycline-related ligand is doxycycline or anhydrotetracycline. Invarious aspects, the repressor is ESR and the sulfonylurea ligand isethametsulfuron, chlorsulfuron, metsulfuron-methyl, sulfometuron methyl,chlorimuron ethyl, nicosulfuron, primisulfuron, tribenuron,sulfosulfuron, trifloxysulfuron, foramsulfuron, iodosulfuron,prosulfuron, thifensulfuron, rimsulfuron, mesosulfuron, or halosulfuron.In various aspects, the nucleotide sequence encoding the WUS/WOXhomeobox polypeptide is operably linked to a constitutive promoter. Invarious aspects, the constitutive promoter is UBI, LLDAV, EVCV, DMMV,BSV (AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO (ALT1),USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO (1.2 KB), IN2-2, NOS, the -135version of 35S, or ZM-ADF PRO (ALT2). In various aspects, the nucleotidesequence encoding the WUS/WOX homeobox polypeptide is operably linked toan inducible promoter or a developmentally-regulated promoter. Invarious aspects, the developmentally-regulated promoter is a PLTPpromoter. In various aspects, the PLTP promoter is derived from amonocot. In various aspects, the monocot is barley, maize, millet, oats,rice, rye, Setaria sp., sorghum, sugarcane, switchgrass, triticale,turfgrass, or wheat. In various aspects, the monocot is maize, sorghum,rice, or Setaria sp. In various aspects, the monocot is maize, sorghumor rice. In various aspects, the monocot is maize. In various aspects,the PLTP promoter is derived from a dicot. In various aspects, the dicotis kale, cauliflower, broccoli, mustard plant, cabbage, pea, clover,alfalfa, broad bean, tomato, cassava, soybean, canola, alfalfa,sunflower, safflower, tobacco. Arabidopsis, or cotton. In variousaspects, the PLTP promoter comprises any one of SEQ ID NO: 1-2 or 55-61.In various aspects, the PLTP promoter comprises any one of SEQ ID NO: 1or 2. In various aspects, the inducible promoter is an auxin-induciblepromoter. In various aspects, the auxin inducible promoter is an AXIG1.In various aspects, the AXIG1 promoter comprises the nucleotide sequenceof SEQ ID NO: 39. In various aspects, the promoter comprises anauxin-response element. In various aspects, the promoter contains one ormore DR5 enhancer motifs. In various aspects, the promoter is a weakconstitutive promoter modified for repression and de-repression. Invarious aspects, one or more operator sequences in the promoter havebeen positioned near or overlapping the TATA box and/or thetranscription start site. In various aspects, the promoter is NOS,AXIG1, ZM-GOS2, CC-UB11-PRO or ZM-ADF4-PRO. In various aspects, thepromoter is a DR5 promoter comprising the nucleotide sequence of SEQ IDNO: 40. In various aspects, the promoter is a de-repressible promoter.In various aspects, the de-repressible promoter is TETR, ESR, or CR. Invarious aspects, the WUS/WOX homeobox polypeptide is a WUS1, WUS2, WUS3,WOX2A, WOX4, WOX5, or WOX9 polypeptide. In various aspects, thenucleotide sequence encoding the WUS/WOX homeobox polypeptide is amonocot nucleotide. In various aspects, the monocot is barley, maize,millet, oats, rice, rye, Setaria sp., sorghum, sugarcane, switchgrass,triticale, turfgrass, or wheat. In various aspects, the nucleotidesequence encoding the WUS/WOX homeobox polypeptide is a dicotnucleotide. In various aspects, the dicot is kale, cauliflower,broccoli, mustard plant, cabbage, pea, clover, alfalfa, broad bean,tomato, cassava, soybean, canola, alfalfa, sunflower, safflower,tobacco, Arabidopsis, or cotton. In various aspects, the nucleotidesequence encoding a WUS/WOX homeobox polypeptide encodes an amino acidsequence comprising any one of SEQ ID NOs:4, 6, 8, 10, 12, 14, or 16. Invarious aspects, the nucleotide sequence encoding a WUS/WOX homeoboxpolypeptide comprises any one of SEQ ID NOs:3, 5, 7, 9, 11, 13, or 15.In various aspects, the WUS/WOX homeobox polypeptide is a WUS1polypeptide. In various aspects, the WUS1 polypeptide is a maize,sorghum, rice or Setaria sp. WUS1 polypeptide. In various aspects, theWUS1 polypeptide is a maize or rice WUS1 polypeptide. In variousaspects, the WUS1 polypeptide is a maize WUS1 polypeptide. In variousaspects, the WUS1 polypeptide comprises an amino acid sequence of SEQ IDNO: 4. In various aspects, the WUS1 polypeptide is encoded by anucleotide sequence comprising SEQ ID NO: 3. In various aspects, theWUS/WOX homeobox polypeptide is a WUS2 polypeptide. In various aspects,the WUS2 polypeptide is a maize, sorghum, rice or Setaria sp. WUS2polypeptide. In various aspects, the WUS2 polypeptide is a maize or riceWUS2 polypeptide. In various aspects, the WUS2 polypeptide is a maizeWUS2 polypeptide. In various aspects, the WUS2 polypeptide comprises theamino acid sequence SEQ ID NO:6. In various aspects, the WUS2polypeptide is encoded by a nucleotide sequence comprising SEQ ID NO: 5.In various aspects, the WUS/WOX homeobox polypeptide is a WUS3polypeptide. In various aspects, the WUS3 polypeptide is a maize,sorghum, rice or Setaria WUS3 polypeptide. In various aspects, the WUS3polypeptide is a maize or rice WUS3 polypeptide. In various aspects, theWUS3 polypeptide is a maize WUS3 polypeptide. In various aspects, theWUS3 polypeptide comprises the amino sequence of SEQ ID NO:8 In variousaspects, the WUS3 polypeptide is encoded by a nucleotide sequencecomprising SEQ ID NO:7. In various aspects, the WUS/WOX homeoboxpolypeptide is a WOX5 polypeptide. In various aspects, the WOX5polypeptide is a WOX5A polypeptide. In various aspects, the WOX5polypeptide is a maize, sorghum, rice or Setaria WOX5 polypeptide. Invarious aspects, the WOX5 polypeptide is a maize or rice WOX5polypeptide. In various aspects, the WOX5 polypeptide is a maize WOX5polypeptide. In various aspects, the WOX5 polypeptide comprises theamino acid sequence of SEQ ID NO: 14. In various aspects, the WOX5polypeptide is encoded by a nucleotide sequence comprising SEQ ID NO:13. In various aspects, the explant is derived from a monocot. Invarious aspects, the monocot is barley, maize, millet, oats, rice, rye,Setaria sp., sorghum, sugarcane, switchgrass, triticale, turfgrass, orwheat. In various aspects, the explant is derived from a dicot. Invarious aspects, the dicot is kale, cauliflower, broccoli, mustardplant, cabbage, pea, clover, alfalfa, broad bean, tomato, cassava,soybean, canola, alfalfa, sunflower, safflower, tobacco, Arabidopsis, orcotton. In various aspects, the regenerable plant structure is formedwithin about 0 to about 7 days of transforming the cell or within about0 to about 14 days of transforming the cell and the transgenic plant isformed in about 14 days of transforming the cell to about 60 days oftransforming the cell. In various aspects, the method is carried out inthe absence of rooting medium. In various aspects, the method is carriedout in the presence of rooting medium. In various aspects, the explantis an immature embryo. In various aspects, the immature embryo is a 1-5mm immature embryo. In various aspects, the immature embryo is a 3.5-5mm immature embryo. In various aspects, exogenous cytokinin is usedduring germinating after about 7 days of transforming the cell or afterabout 14 days of transforming the cell. In various aspects, theexpression of a polypeptide of (a) is transient. In various aspects, aseed from the plant produced by the method is provided.

In a further aspect, the present disclosure provides methods forproducing a transgenic plant, comprising (a) transforming a cell of anexplant with an expression construct comprising a nucleotide sequenceencoding a polypeptide comprising two AP2-DNA binding domains; and (b)allowing expression of the polypeptide of (a) in each transformed cellto form a regenerable plant structure in the absence of exogenouscytokinin, wherein no callus is formed; and (c) germinating theregenerable plant structure to form the transgenic plant. In variousaspects, the expression of the nucleotide sequence encoding thepolypeptide comprising two AP2 binding domains occurs less than 1 day,less than 2 days, less than 5 days, less than 7 days, or less than about14 days after initiation of transformation. In various aspects, theexpression construct further comprises a nucleotide sequence encoding asite-specific recombinase. In various aspects, germinating is performedin the presence of exogenous cytokinin. In various aspects, thesite-specific recombinase is FLP, Cre, SSV1, lambda Int, phi C31 Int,HK022, R, Gin, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, or U153. Invarious aspects, the site-specific recombinase is a destabilized fusionpolypeptide. In various aspects, the destabilized fusion polypeptide isTETR(L17G)˜CRE or ESR(L17G)˜CRE. In various aspects, the nucleotidesequence encoding a site-specific recombinase is operably linked to aconstitutive promoter, an inducible promoter, or adevelopmentally-regulated promoter. In various aspects, the induciblepromoter is GLB1, OLE, LTP2, HSP17.7, HSP26, or HSP18A. In variousaspects, the inducible promoter is a chemically-inducible promoter. Invarious aspects, the chemically-inducible promoter is XVE. In variousaspects, the chemically-inducible promoter is repressed by TETR, ESR, orCR, and de-repression occurs upon addition of tetracycline-related orsulfonylurea ligands. In various aspects, the repressor is TETR and thetetracycline-related ligand is doxycycline or anhydrotetracycline. Invarious aspects, the repressor is ESR and the sulfonylurea ligand isethametsulfuron, chlorsulfuron, metsulfuron-methyl, sulfometuron methyl,chlorimuron ethyl, nicosulfuron, primisulfuron, tribenuron,sulfosulfuron, trifloxysulfuron, foramsulfuron, iodosulfuron,prosulfuron, thifensulfuron, rimsulfuron, mesosulfuron, or halosulfuron.In various aspects, the nucleotide sequence encoding a polypeptidecomprising two AP2-DNA binding domains is operably linked to aninducible promoter or a developmentally-regulated promoter. In variousaspects, the developmentally-regulated promoter is a PLTP promoter. Invarious aspects, the PLTP promoter is derived from a monocot. In variousaspects, the monocot is barley, maize, millet, oats, rice, rye, Setariasp., sorghum, sugarcane, switchgrass, triticale, turfgrass, or wheat. Invarious aspects, the monocot is maize, sorghum, rice, or Setaria sp. Invarious aspects, the monocot is maize, sorghum or rice. In variousaspects, the monocot is maize. In various aspects, the PLTP promoter isderived from a dicot. In various aspects, the dicot is kale,cauliflower, broccoli, mustard plant, cabbage, pea, clover, alfalfa,broad bean, tomato, cassava, soybean, canola, alfalfa, sunflower,safflower, tobacco. Arabidopsis, or cotton. In various aspects, the PLTPpromoter comprises any one of SEQ ID NO: 1-2 or 55-61. In variousaspects, the PLTP promoter comprises any one of SEQ ID NO: 1 or 2. Invarious aspects, the polypeptide comprising the two AP2-DNA bindingdomains is an ODP2, BBM2, BMN2, or BMN3 polypeptide. In various aspects,the polypeptide comprising the two AP2-DNA binding domains is a monocotpolypeptide. In various aspects, the monocot is barley, maize, millet,oats, rice, rye, Setaria sp., sorghum, sugarcane, switchgrass,triticale, turfgrass, or wheat. In various aspects, the monocot ismaize, sorghum, rice, or Setaria sp. In various aspects, the monocot ismaize or rice. In various aspects, the monocot is maize. In variousaspects, the polypeptide comprising the two AP2-DNA binding domains is adicot polypeptide. In various aspects, the dicot is kale, cauliflower,broccoli, mustard plant, cabbage, pea, clover, alfalfa, broad bean,tomato, cassava, soybean, canola, alfalfa, sunflower, safflower,tobacco, Arabidopsis, or cotton. In various aspects, the polypeptidecomprising the two AP2-DNA binding domains comprises an amino acidsequence of any one of SEQ ID NO: 18, 20, 63, 65, or 67. In variousaspects, the polypeptide comprising the two AP2-DNA binding domains isencoded by a nucleotide sequence comprising any one of SEQ ID NO: 17,19, 21, 62, 64, 66, or 68. In various aspects, the polypeptidecomprising the two AP2-DNA binding domains is an ODP2 polypeptide. Invarious aspects, the ODP2 polypeptide is a monocot polypeptide. Invarious aspects, the monocot is barley, maize, millet, oats, rice, rye,Setaria sp., sorghum, sugarcane, switchgrass, triticale, turfgrass, orwheat. In various aspects, the monocot is maize, rice, or Setaria sp. Invarious aspects, the monocot is maize or rice. In various aspects, theODP2 is a dicot polypeptide. In various aspects, the dicot is kale,cauliflower, broccoli, mustard plant, cabbage, pea, clover, alfalfa,broad bean, tomato, cassava, soybean, canola, alfalfa, sunflower,safflower, tobacco, Arabidopsis, or cotton. In various aspects, the ODP2polypeptide comprises the amino acid sequence of any one of SEQ ID NO:18, 63, 65, or 67. In various aspects, the ODP2 polypeptide is encodedby a nucleotide sequence comprising the sequence of any one of SEQ IDNO: 17, 21, 62, 64, 66, or 68. In various aspects, the polypeptidecomprising the two AP2-DNA binding domains is a BBM2 polypeptide. Invarious aspects, the BBM2 polypeptide is a monocot polypeptide. Invarious aspects, the monocot is barley, maize, millet, oats, rice, rye,Setaria sp., sorghum, sugarcane, switchgrass, triticale, turfgrass, orwheat. In various aspects, the monocot is maize, sugarcane, rice, orSetaria sp. In various aspects, the monocot is maize or rice. In variousaspects, the monocot is maize. In various aspects, the BBM2 polypeptideis a dicot polypeptide. In various aspects, the dicot is kale,cauliflower, broccoli, mustard plant, cabbage, pea, clover, alfalfa,broad bean, tomato, cassava, soybean, canola, alfalfa, sunflower,safflower, tobacco, Arabidopsis, or cotton. In various aspects, the BBM2polypeptide comprises the amino acid sequence of SEQ ID NO: 20. Invarious aspects, the BBM2 polypeptide is encoded by a nucleotidesequence comprising the sequence of SEQ ID NO: 19. In various aspects,the explant is derived from a monocot. In various aspects, the monocotis barley, maize, millet, oats, rice, rye, Setaria sp., sorghum,sugarcane, switchgrass, triticale, turfgrass, or wheat. In variousaspects, the explant is derived from a dicot. In various aspects, thedicot is kale, cauliflower, broccoli, mustard plant, cabbage, pea,clover, alfalfa, broad bean, tomato, cassava, soybean, canola, alfalfa,sunflower, safflower, tobacco, Arabidopsis, or cotton. In variousaspects, the regenerable plant structure is formed within about 0 toabout 7 days of transforming the cell or within about 0 to about 14 daysof transforming the cell and the transgenic plant is formed in about 14days of transforming the cell to about 60 days of transforming the cell.In various aspects, the method is carried out in the absence of rootingmedium. In various aspects, the method is carried out in the presence ofrooting medium. In various aspects, the explant is an immature embryo.In various aspects, the immature embryo is a 1-5 mm immature embryo. Invarious aspects, the immature embryo is a 3.5-5 mm immature embryo. Invarious aspects, exogenous cytokinin is used during germinating afterabout 7 days of transforming the cell or after about 14 days oftransforming the cell. In various aspects, the expression of apolypeptide of (a) is transient. In various aspects, a seed from theplant produced by the method is provided.

In a further aspect, the present disclosure provides methods forproducing a transgenic plant, comprising (a) transforming a cell of anexplant with an expression construct comprising (i) a nucleotidesequence encoding a WUS/WOX homeobox polypeptide; and (ii) a nucleotidesequence encoding a polypeptide comprising two AP2-DNA binding domains;(b) allowing expression of the polypeptide of (a) in each transformedcell to form a regenerable plant structure in the absence of exogenouscytokinin, wherein no callus is formed; and (c) germinating theregenerable plant structure to form the transgenic plant. In an aspect,the expression of the nucleotide sequence encoding the WUS/WOX homeoboxpolypeptide and the nucleotide sequence encoding the polypeptidecomprising two AP2 binding domains occurs less than 1 day, less than 2days, less than 5 days, less than 7 days, or less than about 14 daysafter initiation of transformation. In various aspects, germinating isperformed in the presence of exogenous cytokinin. In various aspects,the expression construct further comprises a nucleotide sequenceencoding a site-specific recombinase. In various aspects, thesite-specific recombinase is FLP, Cre, SSV1, lambda Int, phi C31 Int,HK022, R, Gin, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, or U153. Invarious aspects, the site-specific recombinase is a destabilized fusionpolypeptide. In various aspects, the destabilized fusion polypeptide isTETR(L17G)˜CRE or ESR(L17G)˜CRE. In various aspects, the nucleotidesequence encoding a site-specific recombinase is operably linked to aconstitutive promoter, an inducible promoter, or adevelopmentally-regulated promoter. In various aspects, the induciblepromoter is GLB1, OLE, LTP2, HSP17.7, HSP26, or HSP18A. In variousaspects, the inducible promoter is a chemically-inducible promoter. Invarious aspects, the chemically-inducible promoter is XVE. In variousaspects, the chemically-inducible promoter is repressed by TETR, ESR, orCR, and de-repression occurs upon addition of tetracycline-related orsulfonylurea ligands. In various aspects, the repressor is TETR and thetetracycline-related ligand is doxycycline or anhydrotetracycline. Invarious aspects, the repressor is ESR and the sulfonylurea ligand isethametsulfuron, chlorsulfuron, metsulfuron-methyl, sulfometuron methyl,chlorimuron ethyl, nicosulfuron, primisulfuron, tribenuron,sulfosulfuron, trifloxysulfuron, foramsulfuron, iodosulfuron,prosulfuron, thifensulfuron, rimsulfuron, mesosulfuron, or halosulfuron.In various aspects, the nucleotide sequence encoding the WUS/WOXhomeobox polypeptide is operably linked to a constitutive promoter. Invarious aspects, the constitutive promoter is UBI, LLDAV, EVCV, DMMV,BSV (AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO (ALT1),USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO (1.2 KB), IN2-2, NOS, the -135version of 35S, or ZM-ADF PRO (ALT2). In various aspects, the nucleotidesequence encoding the WUS/WOX homeobox polypeptide is operably linked toan inducible promoter or a developmentally-regulated promoter. Invarious aspects, the nucleotide sequence encoding a polypeptidecomprising two AP2-DNA binding domains is operably linked to aninducible promoter or a developmentally-regulated promoter. In variousaspects, the developmentally-regulated promoter is a PLTP promoter. Invarious aspects, the PLTP promoter is derived from a monocot. In variousaspects, the monocot is barley, maize, millet, oats, rice, rye, Setariasp., sorghum, sugarcane, switchgrass, triticale, turfgrass, or wheat. Invarious aspects, the monocot is maize, sorghum, rice, or Setaria sp. Invarious aspects, the monocot is maize, sorghum or rice. In variousaspects, the monocot is maize. In various aspects, the PLTP promoter isderived from a dicot. In various aspects, the dicot is kale,cauliflower, broccoli, mustard plant, cabbage, pea, clover, alfalfa,broad bean, tomato, cassava, soybean, canola, alfalfa, sunflower,safflower, tobacco, Arabidopsis, or cotton. In various aspects, the PLTPpromoter comprises any one of SEQ ID NO: 1-2 or 55-61. In variousaspects, the PLTP promoter comprises any one of SEQ ID NO: 1 or 2. Invarious aspects, the inducible promoter is an auxin-inducible promoter.In various aspects, the auxin inducible promoter is an AXIG1. In variousaspects, the AXIG1 promoter comprises the nucleotide sequence of SEQ IDNO. 39. In various aspects, the promoter comprises an auxin-responseelement. In various aspects, the promoter contains one or more DR5enhancer motifs. In various aspects, the promoter is a weak constitutivepromoter modified for repression and de-repression. In various aspects,one or more operator sequences in the promoter have been positioned nearor overlapping the TATA box and/or the transcription start site. Invarious aspects, the promoter is NOS, AXIG1, ZM-GOS2, CC-UB11-PRO orZM-ADF4-PRO. In various aspects, the promoter is a DR5 promotercomprising the nucleotide sequence of SEQ ID NO: 40. In various aspects,the promoter is a de-repressible promoter. In various aspects, thede-repressible promoter is TETR, ESR, or CR. In various aspects, theWUS/WOX homeobox polypeptide is a WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5,or WOX9 polypeptide. In various aspects, the nucleotide sequenceencoding the WUS/WOX homeobox polypeptide is a monocot nucleotide. Invarious aspects, the monocot is barley, maize, millet, oats, rice, rye,Setaria sp., sorghum, sugarcane, switchgrass, triticale, turfgrass, orwheat. In various aspects, the nucleotide sequence encoding the WUS/WOXhomeobox polypeptide is a dicot nucleotide. In various aspects, thedicot is kale, cauliflower, broccoli, mustard plant, cabbage, pea,clover, alfalfa, broad bean, tomato, cassava, soybean, canola, alfalfa,sunflower, safflower, tobacco. Arabidopsis, or cotton. In variousaspects, the nucleotide sequence encoding a WUS/WOX homeobox polypeptideencodes an amino acid sequence comprising any one of SEQ ID NOs:4, 6, 8,10, 12, 14, or 16. In various aspects, the nucleotide sequence encodinga WUS/WOX homeobox polypeptide comprises any one of SEQ ID NOs:3, 5, 7,9, 11, 13, or 15. In various aspects, the WUS/WOX homeobox polypeptideis a WUS1 polypeptide. In various aspects, the WUS1 polypeptide is amaize, sorghum, rice or Setaria sp. WUS1 polypeptide. In variousaspects, the WUS1 polypeptide is a maize or rice WUS1 polypeptide. Invarious aspects, the WUS1 polypeptide is a maize WUS1 polypeptide. Invarious aspects, the WUS1 polypeptide comprises an amino acid sequenceof SEQ ID NO: 4. In various aspects, the WUS1 polypeptide is encoded bya nucleotide sequence comprising SEQ ID NO: 3. In various aspects, theWUS/WOX homeobox polypeptide is a WUS2 polypeptide. In various aspects,the WUS2 polypeptide is a maize, sorghum, rice or Setaria sp. WUS2polypeptide. In various aspects, the WUS2 polypeptide is a maize or riceWUS2 polypeptide. In various aspects, the WUS2 polypeptide is a maizeWUS2 polypeptide. In various aspects, the WUS2 polypeptide comprises theamino acid sequence SEQ ID NO:6. In various aspects, the WUS2polypeptide is encoded by a nucleotide sequence comprising SEQ ID NO: 5.In various aspects, the WUS/WOX homeobox polypeptide is a WUS3polypeptide. In various aspects, the WUS3 polypeptide is a maize,sorghum, rice or Setaria WUS3 polypeptide. In various aspects, the WUS3polypeptide is a maize or rice WUS3 polypeptide. In various aspects, theWUS3 polypeptide is a maize WUS3 polypeptide. In various aspects, theWUS3 polypeptide comprises the amino sequence of SEQ ID NO:8 In variousaspects, the WUS3 polypeptide is encoded by a nucleotide sequencecomprising SEQ ID NO:7. In various aspects, the WUS/WOX homeoboxpolypeptide is a WOX5 polypeptide. In various aspects, the WOX5polypeptide is a WOX5A polypeptide. In various aspects, the WOX5polypeptide is a maize, sorghum, rice or Setaria WOX5 polypeptide. Invarious aspects, the WOX5 polypeptide is a maize or rice WOX5polypeptide. In various aspects, the WOX5 polypeptide is a maize WOX5polypeptide. In various aspects, the WOX5 polypeptide comprises theamino acid sequence of SEQ ID NO: 14. In various aspects, the WOX5polypeptide is encoded by a nucleotide sequence comprising SEQ ID NO:13. In various aspects, the polypeptide comprising the two AP2-DNAbinding domains is an ODP2, BBM2, BMN2, or BMN3 polypeptide. In variousaspects, the polypeptide comprising the two AP2-DNA binding domains is amonocot polypeptide. In various aspects, the monocot is barley, maize,millet, oats, rice, rye, Setaria sp., sorghum, sugarcane, switchgrass,triticale, turfgrass, or wheat. In various aspects, the monocot ismaize, sorghum, rice, or Setaria sp. In various aspects, the monocot ismaize or rice. In various aspects, the monocot is maize. In variousaspects, the polypeptide comprising the two AP2-DNA binding domains is adicot polypeptide. In various aspects, the dicot is kale, cauliflower,broccoli, mustard plant, cabbage, pea, clover, alfalfa, broad bean,tomato, cassava, soybean, canola, alfalfa, sunflower, safflower,tobacco, Arabidopsis, or cotton. In various aspects, the polypeptidecomprising the two AP2-DNA binding domains comprises an amino acidsequence of any one of SEQ ID NO: 18, 20, 63, 65, or 67. In variousaspects, the polypeptide comprising the two AP2-DNA binding domains isencoded by a nucleotide sequence comprising any one of SEQ ID NO: 17,19, 21, 62, 64, 66, or 68. In various aspects, the polypeptidecomprising the two AP2-DNA binding domains is an ODP2 polypeptide. Invarious aspects, the ODP2 polypeptide is a monocot polypeptide. Invarious aspects, the monocot is barley, maize, millet, oats, rice, rye,Setaria sp., sorghum, sugarcane, switchgrass, triticale, turfgrass, orwheat. In various aspects, the monocot is maize, rice, or Setaria sp. Invarious aspects, the monocot is maize or rice. In various aspects, theODP2 is a dicot polypeptide. In various aspects, the dicot is kale,cauliflower, broccoli, mustard plant, cabbage, pea, clover, alfalfa,broad bean, tomato, cassava, soybean, canola, alfalfa, sunflower,safflower, tobacco, Arabidopsis, or cotton. In various aspects, the ODP2polypeptide comprises the amino acid sequence of any one of SEQ ID NO:18, 63, 65, or 67. In various aspects, the ODP2 polypeptide is encodedby a nucleotide sequence comprising the sequence of any one of SEQ IDNO: 17, 21, 62, 64, 66, or 68. In various aspects, the polypeptidecomprising the two AP2-DNA binding domains is a BBM2 polypeptide. Invarious aspects, the BBM2 polypeptide is a monocot polypeptide. Invarious aspects, the monocot is barley, maize, millet, oats, rice, rye,Setaria sp., sorghum, sugarcane, switchgrass, triticale, turfgrass, orwheat. In various aspects, the monocot is maize, sugarcane, rice, orSetaria sp. In various aspects, the monocot is maize or rice. In variousaspects, the monocot is maize. In various aspects, the BBM2 polypeptideis a dicot polypeptide. In various aspects, the dicot is kale,cauliflower, broccoli, mustard plant, cabbage, pea, clover, alfalfa,broad bean, tomato, cassava, soybean, canola, alfalfa, sunflower,safflower, tobacco, Arabidopsis, or cotton. In various aspects, the BBM2polypeptide comprises the amino acid sequence of SEQ ID NO: 20. Invarious aspects, the BBM2 polypeptide is encoded by a nucleotidesequence comprising the sequence of SEQ ID NO: 19. In various aspects,the explant is derived from a monocot. In various aspects, the monocotis barley, maize, millet, oats, rice, rye, Setaria sp., sorghum,sugarcane, switchgrass, triticale, turfgrass, or wheat. In variousaspects, the explant is derived from a dicot. In various aspects, thedicot is kale, cauliflower, broccoli, mustard plant, cabbage, pea,clover, alfalfa, broad bean, tomato, cassava, soybean, canola, alfalfa,sunflower, safflower, tobacco, Arabidopsis, or cotton. In variousaspects, the regenerable plant structure is formed within about 0 toabout 7 days of transforming the cell or within about 0 to about 14 daysof transforming the cell and the transgenic plant is formed in about 14days of transforming the cell to about 60 days of transforming the cell.In various aspects, the method is carried out in the absence of rootingmedium. In various aspects, the method is carried out in the presence ofrooting medium. In various aspects, the explant is an immature embryo.In various aspects, the immature embryo is a 1-5 mm immature embryo. Invarious aspects, the immature embryo is a 3.5-5 mm immature embryo. Invarious aspects, exogenous cytokinin is used during germinating afterabout 7 days of transforming the cell or after about 14 days oftransforming the cell. In various aspects, the expression of apolypeptide of (a) is transient. In various aspects, a seed from theplant produced by the method is provided.

In a further aspect, the present disclosure provides methods forproducing a transgenic plant, comprising (a) transforming a cell of anexplant with an expression construct comprising (i) a nucleotidesequence encoding a WUS/WOX homeobox polypeptide; or (ii) a nucleotidesequence encoding a polypeptide comprising two AP2-DNA binding domains;or (iii) a combination of (i) and (ii); and (b) allowing expression ofthe polypeptide of (a) in each transformed cell to form a regenerableplant structure in the absence of exogenous cytokinin, wherein no callusis formed; and (c) germinating the regenerable plant structure of (b) toform the transgenic plant in about 14 days to about 60 days. In variousaspects, the expression construct comprises both the nucleotide sequenceencoding the WUS/WOX homeobox polypeptide and the nucleotide sequenceencoding the polypeptide comprising two AP2-DNA binding domains. Invarious aspects, the expression construct comprises the nucleotidesequence encoding the WUS/WOX homeobox polypeptide. In various aspects,the expression construct comprises the nucleotide sequence encoding thepolypeptide comprising two AP2-DNA binding domains. In various aspects,the expression of the nucleotide sequence encoding the WUS/WOX homeoboxpolypeptide and/or the nucleotide sequence encoding the polypeptidecomprising two AP2 binding domains occurs less than 1 day, less than 2days, less than 5 days, less than 7 days, or less than about 14 daysafter initiation of transformation. In various aspects, germinating isperformed in the presence of exogenous cytokinin. In various aspects,the expression construct further comprises a nucleotide sequenceencoding a site-specific recombinase. In various aspects, thesite-specific recombinase is FLP, Cre, SSV1, lambda Int, phi C31 Int,HK022, R, Gin, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, or U153. Invarious aspects, the site-specific recombinase is a destabilized fusionpolypeptide. In various aspects, the destabilized fusion polypeptide isTETR(L17G)˜CRE or ESR(L17G)˜CRE. In various aspects, the nucleotidesequence encoding a site-specific recombinase is operably linked to aconstitutive promoter, an inducible promoter, or adevelopmentally-regulated promoter. In various aspects, the induciblepromoter is GLB1, OLE, LTP2, HSP17.7, HSP26, or HSP18A. In variousaspects, the inducible promoter is a chemically-inducible promoter. Invarious aspects, the chemically-inducible promoter is XVE. In variousaspects, the chemically-inducible promoter is repressed by TETR, ESR, orCR, and de-repression occurs upon addition of tetracycline-related orsulfonylurea ligands. In various aspects, the repressor is TETR and thetetracycline-related ligand is doxycycline or anhydrotetracycline. Invarious aspects, the repressor is ESR and the sulfonylurea ligand isethametsulfuron, chlorsulfuron, metsulfuron-methyl, sulfometuron methyl,chlorimuron ethyl, nicosulfuron, primisulfuron, tribenuron,sulfosulfuron, trifloxysulfuron, foramsulfuron, iodosulfuron,prosulfuron, thifensulfuron, rimsulfuron, mesosulfuron, or halosulfuron.In various aspects, the nucleotide sequence encoding the WUS/WOXhomeobox polypeptide is operably linked to a constitutive promoter. Invarious aspects, the constitutive promoter is UBI, LLDAV, EVCV, DMMV,BSV (AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO (ALT1),USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO (1.2 KB), IN2-2, NOS, the -135version of 35S, or ZM-ADF PRO (ALT2). In various aspects, the nucleotidesequence encoding the WUS/WOX homeobox polypeptide is operably linked toan inducible promoter or a developmentally-regulated promoter. Invarious aspects, the nucleotide sequence encoding a polypeptidecomprising two AP2-DNA binding domains is operably linked to aninducible promoter or a developmentally-regulated promoter. In variousaspects, the developmentally-regulated promoter is a PLTP promoter. Invarious aspects, the PLTP promoter is derived from a monocot. In variousaspects, the monocot is barley, maize, millet, oats, rice, rye, Setariasp., sorghum, sugarcane, switchgrass, triticale, turfgrass, or wheat. Invarious aspects, the monocot is maize, sorghum, rice, or Setaria sp. Invarious aspects, the monocot is maize, sorghum or rice. In variousaspects, the monocot is maize. In various aspects, the PLTP promoter isderived from a dicot. In various aspects, the dicot is kale,cauliflower, broccoli, mustard plant, cabbage, pea, clover, alfalfa,broad bean, tomato, cassava, soybean, canola, alfalfa, sunflower,safflower, tobacco, Arabidopsis, or cotton. In various aspects, the PLTPpromoter comprises any one of SEQ ID NO: 1-2 or 55-61. In variousaspects, the PLTP promoter comprises any one of SEQ ID NO: 1 or 2. Invarious aspects, the inducible promoter is an auxin-inducible promoter.In various aspects, the auxin inducible promoter is an AXIG1. In variousaspects, the AXIG1 promoter comprises the nucleotide sequence of SEQ IDNO: 39. In various aspects, the promoter comprises an auxin-responseelement. In various aspects, the promoter contains one or more DR5enhancer motifs. In various aspects, the promoter is a weak constitutivepromoter modified for repression and de-repression. In various aspects,one or more operator sequences in the promoter have been positioned nearor overlapping the TATA box and/or the transcription start site. Invarious aspects, the promoter is NOS, AXIG1, ZM-GOS2, CC-UB11-PRO orZM-ADF4-PRO. In various aspects, the promoter is a DR5 promotercomprising the nucleotide sequence of SEQ ID NO: 40. In various aspects,the promoter is a de-repressible promoter. In various aspects, thede-repressible promoter is TETR, ESR, or CR. In various aspects, theWUS/WOX homeobox polypeptide is a WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5,or WOX9 polypeptide. In various aspects, the nucleotide sequenceencoding the WUS/WOX homeobox polypeptide is a monocot nucleotide. Invarious aspects, the monocot is barley, maize, millet, oats, rice, rye,Setaria sp., sorghum, sugarcane, switchgrass, triticale, turfgrass, orwheat. In various aspects, the nucleotide sequence encoding the WUS/WOXhomeobox polypeptide is a dicot nucleotide. In various aspects, thedicot is kale, cauliflower, broccoli, mustard plant, cabbage, pea,clover, alfalfa, broad bean, tomato, cassava, soybean, canola, alfalfa,sunflower, safflower, tobacco, Arabidopsis, or cotton. In variousaspects, the nucleotide sequence encoding a WUS/WOX homeobox polypeptideencodes an amino acid sequence comprising any one of SEQ ID NOs:4, 6, 8,10, 12, 14, or 16. In various aspects, the nucleotide sequence encodinga WUS/WOX homeobox polypeptide comprises any one of SEQ ID NOs:3, 5, 7,9, 11, 13, or 15. In various aspects, the WUS/WOX homeobox polypeptideis a WUS1 polypeptide. In various aspects, the WUS1 polypeptide is amaize, sorghum, rice or Setaria sp. WUS1 polypeptide. In variousaspects, the WUS1 polypeptide is a maize or rice WUS1 polypeptide. Invarious aspects, the WUS1 polypeptide is a maize WUS1 polypeptide. Invarious aspects, the WUS1 polypeptide comprises an amino acid sequenceof SEQ ID NO: 4. In various aspects, the WUS1 polypeptide is encoded bya nucleotide sequence comprising SEQ ID NO: 3. In various aspects, theWUS/WOX homeobox polypeptide is a WUS2 polypeptide. In various aspects,the WUS2 polypeptide is a maize, sorghum, rice or Setaria sp. WUS2polypeptide. In various aspects, the WUS2 polypeptide is a maize or riceWUS2 polypeptide. In various aspects, the WUS2 polypeptide is a maizeWUS2 polypeptide. In various aspects, the WUS2 polypeptide comprises theamino acid sequence SEQ ID NO:6. In various aspects, the WUS2polypeptide is encoded by a nucleotide sequence comprising SEQ ID NO: 5.In various aspects, the WUS/WOX homeobox polypeptide is a WUS3polypeptide. In various aspects, the WUS3 polypeptide is a maize,sorghum, rice or Setaria WUS3 polypeptide. In various aspects, the WUS3polypeptide is a maize or rice WUS3 polypeptide. In various aspects, theWUS3 polypeptide is a maize WUS3 polypeptide. In various aspects, theWUS3 polypeptide comprises the amino sequence of SEQ ID NO:8. In variousaspects, the WUS3 polypeptide is encoded by a nucleotide sequencecomprising SEQ ID NO:7. In various aspects, the WUS/WOX homeoboxpolypeptide is a WOX5 polypeptide. In various aspects, the WOX5polypeptide is a WOX5A polypeptide. In various aspects, the WOX5polypeptide is a maize, sorghum, rice or Setaria WOX5 polypeptide. Invarious aspects, the WOX5 polypeptide is a maize or rice WOX5polypeptide. In various aspects, the WOX5 polypeptide is a maize WOX5polypeptide. In various aspects, the WOX5 polypeptide comprises theamino acid sequence of SEQ ID NO: 14. In various aspects, the WOX5polypeptide is encoded by a nucleotide sequence comprising SEQ ID NO:13. In various aspects, the polypeptide comprising the two AP2-DNAbinding domains is an ODP2, BBM2, BMN2, or BMN3 polypeptide. In variousaspects, the polypeptide comprising the two AP2-DNA binding domains is amonocot polypeptide. In various aspects, the monocot is barley, maize,millet, oats, rice, rye, Setaria sp., sorghum, sugarcane, switchgrass,triticale, turfgrass, or wheat. In various aspects, the monocot ismaize, sorghum, rice, or Setaria sp. In various aspects, the monocot ismaize or rice. In various aspects, the monocot is maize. In variousaspects, the polypeptide comprising the two AP2-DNA binding domains is adicot polypeptide. In various aspects, the dicot is kale, cauliflower,broccoli, mustard plant, cabbage, pea, clover, alfalfa, broad bean,tomato, cassava, soybean, canola, alfalfa, sunflower, safflower,tobacco, Arabidopsis, or cotton. In various aspects, the polypeptidecomprising the two AP2-DNA binding domains comprises an amino acidsequence of any one of SEQ ID NO: 18, 20, 63, 65, or 67. In variousaspects, the polypeptide comprising the two AP2-DNA binding domains isencoded by a nucleotide sequence comprising any one of SEQ ID NO: 17,19, 21, 62, 64, 66, or 68. In various aspects, the polypeptidecomprising the two AP2-DNA binding domains is an ODP2 polypeptide. Invarious aspects, the ODP2 polypeptide is a monocot polypeptide. Invarious aspects, the monocot is barley, maize, millet, oats, rice, rye.Setaria sp., sorghum, sugarcane, switchgrass, triticale, turfgrass, orwheat. In various aspects, the monocot is maize, rice, or Setaria sp. Invarious aspects, the monocot is maize or rice. In various aspects, theODP2 is a dicot polypeptide. In various aspects, the dicot is kale,cauliflower, broccoli, mustard plant, cabbage, pea, clover, alfalfa,broad bean, tomato, cassava, soybean, canola, alfalfa, sunflower,safflower, tobacco, Arabidopsis, or cotton. In various aspects, the ODP2polypeptide comprises the amino acid sequence of any one of SEQ ID NO:18, 63, 65, or 67. In various aspects, the ODP2 polypeptide is encodedby a nucleotide sequence comprising the sequence of any one of SEQ IDNO: 17, 21, 62, 64, 66, or 68. In various aspects, the polypeptidecomprising the two AP2-DNA binding domains is a BBM2 polypeptide. Invarious aspects, the BBM2 polypeptide is a monocot polypeptide. Invarious aspects, the monocot is barley, maize, millet, oats, rice, rye,Setaria sp., sorghum, sugarcane, switchgrass, triticale, turfgrass, orwheat. In various aspects, the monocot is maize, sugarcane, rice, orSetaria sp. In various aspects, the monocot is maize or rice. In variousaspects, the monocot is maize. In various aspects, the BBM2 polypeptideis a dicot polypeptide. In various aspects, the dicot is kale,cauliflower, broccoli, mustard plant, cabbage, pea, clover, alfalfa,broad bean, tomato, cassava, soybean, canola, alfalfa, sunflower,safflower, tobacco, Arabidopsis, or cotton. In various aspects, the BBM2polypeptide comprises the amino acid sequence of SEQ ID NO: 20. Invarious aspects, the BBM2 polypeptide is encoded by a nucleotidesequence comprising the sequence of SEQ ID NO: 19. In various aspects,the explant is derived from a monocot. In various aspects, the monocotis barley, maize, millet, oats, rice, rye, Setaria sp., sorghum,sugarcane, switchgrass, triticale, turfgrass, or wheat. In variousaspects, the explant is derived from a dicot. In various aspects, thedicot is kale, cauliflower, broccoli, mustard plant, cabbage, pea,clover, alfalfa, broad bean, tomato, cassava, soybean, canola, alfalfa,sunflower, safflower, tobacco, Arabidopsis, or cotton. In variousaspects, the regenerable plant structure is formed within about 0 toabout 7 days of transforming the cell or within about 0 to about 14 daysof transforming the cell. In various aspects, the method is carried outin the absence of rooting medium. In various aspects, the method iscarried out in the presence of rooting medium. In various aspects, theexplant is an immature embryo. In various aspects, the immature embryois a 1-5 mm immature embryo. In various aspects, the immature embryo isa 3.5-5 mm immature embryo. In various aspects, exogenous cytokinin isused during germinating after about 7 days of transforming the cell orafter about 14 days of transforming the cell. In various aspects, theexpression of a polypeptide of (a) is transient. In various aspects, aseed from the plant produced by the method is provided.

In a further aspect, the present disclosure provides methods forproducing a transgenic plant, comprising (a) transforming a cell of anexplant with an expression construct comprising (i) a nucleotidesequence encoding a WUS/WOX homeobox polypeptide; or (ii) a nucleotidesequence encoding a polypeptide comprising two AP2-DNA binding domains;or (iii) a combination of (i) and (ii); and (b) allowing expression ofthe polypeptide of (a) in each transformed cell to form a regenerableplant structure in the absence of exogenous cytokinin, wherein no callusis formed and wherein the regenerable plant structure is formed withinabout 0-7 days or about 0-14 days of transforming the cell; and (c)germinating the regenerable plant structure of (b) to form thetransgenic plant in about 14 days to about 60 days. In various aspects,the expression construct comprises both the nucleotide sequence encodingthe WUS/WOX homeobox polypeptide and the nucleotide sequence encodingthe polypeptide comprising two AP2-DNA binding domains. In variousaspects, the expression construct comprises the nucleotide sequenceencoding the WUS/WOX homeobox polypeptide. In various aspects, theexpression construct comprises the nucleotide sequence encoding thepolypeptide comprising two AP2-DNA binding domains. In various aspects,the expression of the nucleotide sequence encoding the WUS/WOX homeoboxpolypeptide and/or the nucleotide sequence encoding the polypeptidecomprising two AP2 binding domains occurs less than 1 day, less than 2days, less than 5 days, less than 7 days, or less than about 14 daysafter initiation of transformation. In various aspects, germinating isperformed in the presence of exogenous cytokinin. In various aspects,the expression construct further comprises a nucleotide sequenceencoding a site-specific recombinase. In various aspects, thesite-specific recombinase is FLP, Cre, SSV1, lambda Int, phi C31 Int,HK022. R. Gin, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, or U153. Invarious aspects, the site-specific recombinase is a destabilized fusionpolypeptide. In various aspects, the destabilized fusion polypeptide isTETR(L17G)˜CRE or ESR(L17G)˜CRE. In various aspects, the nucleotidesequence encoding a site-specific recombinase is operably linked to aconstitutive promoter, an inducible promoter, or adevelopmentally-regulated promoter. In various aspects, the induciblepromoter is GLB1, OLE, LTP2, HSP17.7, HSP26, or HSP18A. In variousaspects, the inducible promoter is a chemically-inducible promoter. Invarious aspects, the chemically-inducible promoter is XVE. In variousaspects, the chemically-inducible promoter is repressed by TETR, ESR, orCR, and de-repression occurs upon addition of tetracycline-related orsulfonylurea ligands. In various aspects, the repressor is TETR and thetetracycline-related ligand is doxycycline or anhydrotetracycline. Invarious aspects, the repressor is ESR and the sulfonylurea ligand isethametsulfuron, chlorsulfuron, metsulfuron-methyl, sulfometuron methyl,chlorimuron ethyl, nicosulfuron, primisulfuron, tribenuron,sulfosulfuron, trifloxysulfuron, foramsulfuron, iodosulfuron,prosulfuron, thifensulfuron, rimsulfuron, mesosulfuron, or halosulfuron.In various aspects, the nucleotide sequence encoding the WUS/WOXhomeobox polypeptide is operably linked to a constitutive promoter. Invarious aspects, the constitutive promoter is UBI, LLDAV, EVCV, DMMV,BSV (AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO (ALT1),USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO (1.2 KB), IN2-2, NOS, the -135version of 35S, or ZM-ADF PRO (ALT2). In various aspects, the nucleotidesequence encoding the WUS/WOX homeobox polypeptide is operably linked toan inducible promoter or a developmentally-regulated promoter. Invarious aspects, the nucleotide sequence encoding a polypeptidecomprising two AP2-DNA binding domains is operably linked to aninducible promoter or a developmentally-regulated promoter. In variousaspects, the developmentally-regulated promoter is a PLTP promoter. Invarious aspects, the PLTP promoter is derived from a monocot. In variousaspects, the monocot is barley, maize, millet, oats, rice, rye, Setariasp., sorghum, sugarcane, switchgrass, triticale, turfgrass, or wheat. Invarious aspects, the monocot is maize, sorghum, rice, or Setaria sp. Invarious aspects, the monocot is maize, sorghum or rice. In variousaspects, the monocot is maize. In various aspects, the PLTP promoter isderived from a dicot. In various aspects, the dicot is kale,cauliflower, broccoli, mustard plant, cabbage, pea, clover, alfalfa,broad bean, tomato, cassava, soybean, canola, alfalfa, sunflower,safflower, tobacco, Arabidopsis, or cotton. In various aspects, the PLTPpromoter comprises any one of SEQ ID NO: 1-2 or 55-61. In variousaspects, the PLTP promoter comprises any one of SEQ ID NO: 1 or 2. Invarious aspects, the inducible promoter is an auxin-inducible promoter.In various aspects, the auxin inducible promoter is an AXIG1. In variousaspects, the AXIG1 promoter comprises the nucleotide sequence of SEQ IDNO: 39. In various aspects, the promoter comprises an auxin-responseelement. In various aspects, the promoter contains one or more DR5enhancer motifs. In various aspects, the promoter is a weak constitutivepromoter modified for repression and de-repression. In various aspects,one or more operator sequences in the promoter have been positioned nearor overlapping the TATA box and/or the transcription start site. Invarious aspects, the promoter is NOS, AXIG1, ZM-GOS2, CC-UB11-PRO orZM-ADF4-PRO. In various aspects, the promoter is a DR5 promotercomprising the nucleotide sequence of SEQ ID NO: 40. In various aspects,the promoter is a de-repressible promoter. In various aspects, thede-repressible promoter is TETR, ESR, or CR. In various aspects, theWUS/WOX homeobox polypeptide is a WUS1, WUS2, WUS3, WOX2A, WOX4, WOX5,or WOX9 polypeptide. In various aspects, the nucleotide sequenceencoding the WUS/WOX homeobox polypeptide is a monocot nucleotide. Invarious aspects, the monocot is barley, maize, millet, oats, rice, rye,Setaria sp., sorghum, sugarcane, switchgrass, triticale, turfgrass, orwheat. In various aspects, the nucleotide sequence encoding the WUS/WOXhomeobox polypeptide is a dicot nucleotide. In various aspects, thedicot is kale, cauliflower, broccoli, mustard plant, cabbage, pea,clover, alfalfa, broad bean, tomato, cassava, soybean, canola, alfalfa,sunflower, safflower, tobacco, Arabidopsis, or cotton. In variousaspects, the nucleotide sequence encoding a WUS/WOX homeobox polypeptideencodes an amino acid sequence comprising any one of SEQ ID NOs:4, 6, 8,10, 12, 14, or 16. In various aspects, the nucleotide sequence encodinga WUS/WOX homeobox polypeptide comprises any one of SEQ ID NOs:3, 5, 7,9, 11, 13, or 15. In various aspects, the WUS/WOX homeobox polypeptideis a WUS1 polypeptide. In various aspects, the WUS1 polypeptide is amaize, sorghum, rice or Setaria sp. WUS1 polypeptide. In variousaspects, the WUS1 polypeptide is a maize or rice WUS1 polypeptide. Invarious aspects, the WUS1 polypeptide is a maize WUS1 polypeptide. Invarious aspects, the WUS1 polypeptide comprises an amino acid sequenceof SEQ ID NO: 4. In various aspects, the WUS1 polypeptide is encoded bya nucleotide sequence comprising SEQ ID NO: 3. In various aspects, theWUS/WOX homeobox polypeptide is a WUS2 polypeptide. In various aspects,the WUS2 polypeptide is a maize, sorghum, rice or Setaria sp. WUS2polypeptide. In various aspects, the WUS2 polypeptide is a maize or riceWUS2 polypeptide. In various aspects, the WUS2 polypeptide is a maizeWUS2 polypeptide. In various aspects, the WUS2 polypeptide comprises theamino acid sequence SEQ ID NO:6. In various aspects, the WUS2polypeptide is encoded by a nucleotide sequence comprising SEQ ID NO: 5.In various aspects, the WUS/WOX homeobox polypeptide is a WUS3polypeptide. In various aspects, the WUS3 polypeptide is a maize,sorghum, rice or Setaria WUS3 polypeptide. In various aspects, the WUS3polypeptide is a maize or rice WUS3 polypeptide. In various aspects, theWUS3 polypeptide is a maize WUS3 polypeptide. In various aspects, theWUS3 polypeptide comprises the amino sequence of SEQ ID NO:8 In variousaspects, the WUS3 polypeptide is encoded by a nucleotide sequencecomprising SEQ ID NO:7. In various aspects, the WUS/WOX homeoboxpolypeptide is a WOX5 polypeptide. In various aspects, the WOX5polypeptide is a WOX5A polypeptide. In various aspects, the WOX5polypeptide is a maize, sorghum, rice or Setaria WOX5 polypeptide. Invarious aspects, the WOX5 polypeptide is a maize or rice WOX5polypeptide. In various aspects, the WOX5 polypeptide is a maize WOX5polypeptide. In various aspects, the WOX5 polypeptide comprises theamino acid sequence of SEQ ID NO: 14. In various aspects, the WOX5polypeptide is encoded by a nucleotide sequence comprising SEQ ID NO:13. In various aspects, the polypeptide comprising the two AP2-DNAbinding domains is an ODP2, BBM2, BMN2, or BMN3 polypeptide. In variousaspects, the polypeptide comprising the two AP2-DNA binding domains is amonocot polypeptide. In various aspects, the monocot is barley, maize,millet, oats, rice, rye, Setaria sp., sorghum, sugarcane, switchgrass,triticale, turfgrass, or wheat. In various aspects, the monocot ismaize, sorghum, rice, or Setaria sp. In various aspects, the monocot ismaize or rice. In various aspects, the monocot is maize. In variousaspects, the polypeptide comprising the two AP2-DNA binding domains is adicot polypeptide. In various aspects, the dicot is kale, cauliflower,broccoli, mustard plant, cabbage, pea, clover, alfalfa, broad bean,tomato, cassava, soybean, canola, alfalfa, sunflower, safflower,tobacco, Arabidopsis, or cotton. In various aspects, the polypeptidecomprising the two AP2-DNA binding domains comprises an amino acidsequence of any one of SEQ ID NO: 18, 20, 63, 65, or 67. In variousaspects, the polypeptide comprising the two AP2-DNA binding domains isencoded by a nucleotide sequence comprising any one of SEQ ID NO: 17,19, 21, 62, 64, 66, or 68. In various aspects, the polypeptidecomprising the two AP2-DNA binding domains is an ODP2 polypeptide. Invarious aspects, the ODP2 polypeptide is a monocot polypeptide. Invarious aspects, the monocot is barley, maize, millet, oats, rice, rye,Setaria sp., sorghum, sugarcane, switchgrass, triticale, turfgrass, orwheat. In various aspects, the monocot is maize, rice, or Setaria sp. Invarious aspects, the monocot is maize or rice. In various aspects, theODP2 is a dicot polypeptide. In various aspects, the dicot is kale,cauliflower, broccoli, mustard plant, cabbage, pea, clover, alfalfa,broad bean, tomato, cassava, soybean, canola, alfalfa, sunflower,safflower, tobacco, Arabidopsis, or cotton. In various aspects, the ODP2polypeptide comprises the amino acid sequence of any one of SEQ ID NO:18, 63, 65, or 67. In various aspects, the ODP2 polypeptide is encodedby a nucleotide sequence comprising the sequence of any one of SEQ IDNO: 17, 21, 62, 64, 66, or 68. In various aspects, the polypeptidecomprising the two AP2-DNA binding domains is a BBM2 polypeptide. Invarious aspects, the BBM2 polypeptide is a monocot polypeptide. Invarious aspects, the monocot is barley, maize, millet, oats, rice, rye,Setaria sp., sorghum, sugarcane, switchgrass, triticale, turfgrass, orwheat. In various aspects, the monocot is maize, sugarcane, rice, orSetaria sp. In various aspects, the monocot is maize or rice. In variousaspects, the monocot is maize. In various aspects, the BBM2 polypeptideis a dicot polypeptide. In various aspects, the dicot is kale,cauliflower, broccoli, mustard plant, cabbage, pea, clover, alfalfa,broad bean, tomato, cassava, soybean, canola, alfalfa, sunflower,safflower, tobacco, Arabidopsis, or cotton. In various aspects, the BBM2polypeptide comprises the amino acid sequence of SEQ ID NO: 20. Invarious aspects, the BBM2 polypeptide is encoded by a nucleotidesequence comprising the sequence of SEQ ID NO: 19. In various aspects,the explant is derived from a monocot. In various aspects, the monocotis barley, maize, millet, oats, rice, rye, Setaria sp., sorghum,sugarcane, switchgrass, triticale, turfgrass, or wheat. In variousaspects, the explant is derived from a dicot. In various aspects, thedicot is kale, cauliflower, broccoli, mustard plant, cabbage, pea,clover, alfalfa, broad bean, tomato, cassava, soybean, canola, alfalfa,sunflower, safflower, tobacco, Arabidopsis, or cotton. In variousaspects, the method is carried out in the absence of rooting medium. Invarious aspects, the method is carried out in the presence of rootingmedium. In various aspects, the explant is an immature embryo. Invarious aspects, the immature embryo is a 1-5 mm immature embryo. Invarious aspects, the immature embryo is a 3.5-5 mm immature embryo. Invarious aspects, exogenous cytokinin is used during germinating afterabout 7 days of transforming the cell or after about 14 days oftransforming the cell. In various aspects, the expression of apolypeptide of (a) is transient. In various aspects, a seed from theplant produced by the method is provided.

In an aspect, the present disclosure further provides kits comprising(a) an expression construct comprising (i) a nucleotide sequenceencoding a WUS/WOX homeobox polypeptide; or (ii) a nucleotide sequenceencoding a polypeptide comprising two AP2-DNA binding domains; or (iii)a combination of (i) and (ii); and (b) instructions for obtaining aplant regenerable structure in the absence of exogenous cytokinin,wherein no callus is formed. In various aspects, the kit providesinstructions for obtaining a transformed plant within about 14 days toabout 60 days. In various aspects, the kit provides instructions forobtaining a plant from an explant transformed using the kit.

In an aspect, the present disclosure further provides kits comprising(a) an expression construct comprising a nucleotide sequence encoding aWUS/WOX homeobox polypeptide; and (b) instructions for obtaining a plantregenerable structure in the absence of exogenous cytokinin, wherein nocallus is formed. In various aspects, the kit provides instructions forobtaining a transformed plant within about 14 days to about 60 days. Invarious aspects, the kit provides instructions for obtaining a plantfrom an explant transformed using the kit.

In an aspect, the present disclosure further provides kits comprising(a) an expression construct comprising a nucleotide sequence encoding apolypeptide comprising two AP2-DNA binding domains; or (iii) acombination of (i) and (ii); and (b) instructions for obtaining a plantregenerable structure in the absence of exogenous cytokinin, wherein nocallus is formed. In various aspects, the kit provides instructions forobtaining a transformed plant within about 14 days to about 60 days. Invarious aspects, the kit provides instructions for obtaining a plantfrom an explant transformed using the kit.

In a further aspect, the present disclosure provides a seed from theplant produced from the methods disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows a representative image of an immature embryo treated usingthe disclosed methods (see Example 5). The arrows indicaterepresentative newly formed embryos on the surface following treatment.

FIG. 2 shows a representative image of an immature embryo treated usingthe disclosed methods (see Example 5). The image shows fluorescentembryos on the surface following treatment.

FIG. 3 shows a representative image of multiple mature somatic embryosderived from a single original zygotic immature embryo obtained usingthe disclosed methods (see Example 7) after growth on maturation mediumfor 13 days.

FIG. 4 shows a representative image of plants obtained from 45originally-transformed zygotic immature embryos using the disclosedmethods (see Example 7).

FIG. 5 shows early germinating shoots derived from rapidly-formedsomatic embryos two weeks after Agrobacterium infection of immatureembryos of Pioneer elite Spring wheat variety HC0456D.

FIG. 6 shows a representative image of early anticlinal divisions on thesurface of the scutellum (arrows) from an immature zygotic embryo twodays after Agrobacterium transfection.

FIG. 7 shows a representative image of a small globular somatic embryoforming on the surface of the scutellum from a transfected immaturezygotic embryo two days after Agrobacterium transfection.

FIG. 8 shows a representative image of a larger globular somatic embryoforming on the surface of the scutellum from the transfected immaturezygotic embryo two days after Agrobacterium transfection.

FIG. 9 shows a representative image of a transition-stage somatic embryothat formed on the surface of the scutellum from the transfectedimmature zygotic embryo four days after Agrobacterium transfection,showing the smooth surface of the somatic embryo and the lack ofvascular connections with the underlying zygotic embryo.

FIG. 10 shows a representative image of multiple transition-stagesomatic embryos that have formed on the surface of the scutellum fromthe transfected immature zygotic embryo four days after Agrobacteriumtransfection, demonstrating that somatic embryos in close proximity werederived from the underlying zygotic embryo surface and not throughsecondary somatic embryogenesis.

FIG. 11 shows a representative higher magnification image demonstratingthe high mitotic index within the developing somatic embryos, asindicated by the metaphase and anaphase structures observed in multiplecells within the field of view (as indicated by the arrows).

FIG. 12 shows a maize immature embryo 10 days after being transformedwith a T-DNA containing AXIG1 PRO::WUS2 with PLTP PRO::BBM. Numeroussomatic embryos, some of which have fused together as they developed,and an early developing leaf are observed growing from the scutellumsurface. After staining for 20 minutes with Oil Red O, and washing twicein 70% isoproponol, the lipids that accumulated during somatic embryodevelopment stained red (black arrows), while the developing leaf(labeled with a white arrow) and the originally-transformed zygoticscutellum (labeled with a white arrow) accumulated very little stainindicating the typical lower level of lipid deposition in these tissues.

FIG. 13 shows a light micrograph of somatic embryo cells after theembryo was pressed between a cover slip and slide and then stained withOil Red O. Red-staining lipid droplets (as exemplified by the blackarrows) were clearly observed scattered within the cells.

FIG. 14 shows Illumina transcript quantitation for a representativeconstitutively-expressed soy EF1 A gene being expressed across all theplant tissues sampled.

FIG. 15 shows Illumina transcript quantitation for theembryo/seed-specific gene LTP3, the respective transcript levels beingindicative of promoter activity in various plant organs and differentdevelopmental stages.

FIG. 16 shows transformation response as measured by the frequency oftreated immature cotyledons that produced somatic embryos afterAgrobacterium-mediated transformation to introduce a T-DNA containing anexpression cassette with the Arabidopsis WUS gene behind one of fivepromoters; Gm-Phytochrome P450 promoter (P450 PRO); Gm-GlycosylHydrolase promoter (GH PRO); Gm-Homeodomain/Start-domain proteinpromoter (HSD PRO); Gm-LTP3 promoter (LTP3 PRO); Gm-StrictosidineSynthase-Like1 promoter (SSL1 PRO); the negative control with no WUSexpression (NEG CON). For each promoter, the upper and lower ends of thebox indicate the upper and lower quartile for the distribution of thedata, while the line within the box represents the median. For the P450PRO only two replicates were included in this analysis and thus nomedian was calculated.

FIG. 17A shows a light micrograph and FIG. 17B shows the correspondingepifluorescence image of somatic embryos that were moved onto maturationmedium to complete embryo development (shown on maturation medium 35days after the underlying immature cotyledon was transformed with aT-DNA containing Gm-LTP3 PRO::At-WUS). Arrow points to one of the redfluorescing somatic cotyledons; the scale bars represent 2 mm in length.

DETAILED DESCRIPTION

The disclosures herein will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allpossible aspects are shown. Indeed, disclosures may be embodied in manydifferent forms and should not be construed as limited to the aspectsset forth herein; rather, these aspects are provided so that thisdisclosure will satisfy applicable legal requirements.

Many modifications and other aspects disclosed herein will come to mindto one skilled in the art to which the disclosed compositions andmethods pertain having the benefit of the teachings presented in thefollowing descriptions and the associated drawings. Therefore, it is tobe understood that the disclosures are not to be limited to the specificaspects disclosed and that modifications and other aspects are intendedto be included within the scope of the appended claims. Althoughspecific terms are employed herein, they are used in a generic anddescriptive sense only and not for purposes of limitation.

It is also to be understood that the terminology used herein is for thepurpose of describing particular aspects only and is not intended to belimiting. As used in the specification and in the claims, the term“comprising” can include the aspect of “consisting of.” Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich the disclosed compositions and methods belong. In thisspecification and in the claims which follow, reference will be made toa number of terms which shall be defined herein.

I. Methods and Compositions for Producing Transgenic Plants

The present disclosure comprises methods and compositions for producinga transgenic plant, comprising (a) transforming a cell of an explantwith an expression construct comprising: (i) a nucleotide sequenceencoding a WUS/WOX homeobox polypeptide; (ii) a nucleotide sequenceencoding a polypeptide comprising two AP2-DNA binding domains; or (iii)a combination of (i) and (ii); and (b) allowing expression of thepolypeptide of (a) in each transformed cell to form a regenerable plantstructure in the absence of exogenous cytokinin, wherein no callus isformed; and (c) germinating the regenerable plant structure to form thetransgenic plant. The regenerable plant structure is produced withinabout 0-7 days or about 0-14 days of transformation. In an aspect, thegerminating comprises transferring the regenerable plant structure to amaturation medium comprising an exogenous cytokinin and forming thetransgenic plant. In an aspect, the nucleotide sequence encoding aWUS/WOX homeobox polypeptide is capable of stimulating formation of aregenerable plant structure. In an aspect, the nucleotide sequenceencoding a polypeptide comprising two AP2-DNA binding domains is capableof stimulating formation of a regenerable plant structure.

In an aspect, expression of the polypeptide of (a) in each transformedcell of the method is controlled. The controlled expression is pulsedfor a particular period of time. The control of expression of thepolypeptide of (a) can be achieved by a variety of methods as disclosedherein below.

It is to be understood that the methods of the present disclosureproduce single regenerable plant structures that form directly from asingle cell on, or within, the explant that has been transformed, withno intervening single-cell-derived cell or tissue proliferationoccurring before initiation of the regenerable plant structure. Incontrast, previously described methods of transformation andregeneration known in the art involve intervening single-cell-derivedcell- or tissue-proliferation, which comprise types of growth patternsreferred to as callus, non-differentiated callus, embryogenic callus andorganogenic callus. In various aspects, the term “callus” refers to anundifferentiated cell clump under uncontrolled growth. A callus can beobtained by culturing a differentiated cell of a plant tissue in amedium containing a plant growth regulator such as auxin (e.g., 2,4-D)or cytokinin (wherein the medium with cytokinin is referred to asdedifferentiation medium). Examples of auxins that can be added totissue culture medium to stimulate embryogenesis include the naturallyoccurring auxins such as indole-3-acetic acid (IAA),chloroindole-3-acetic acid (CL-IAA), 2-phenylacetic acid (PAA),indole-3-propionic acid (IPA) and indole-3-buteric acid (IBA), inaddition to synthetic auxins such as 1-naphthaleneacetic acid (NAA),2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyaceticacid (2,4,5-T). Examples of cytokinins that can be added to tissueculture medium to stimulate morphogenesis (and in particular meristemand shoot development) include benzylaminopurine (BAP), zeatin, kinetin,thidiazuron (TDZ), meta-topolin, and methoxy-topolins. Auxins andcytokinins which are added to tissue culture medium are exogenous. It isto be further understood that previously described methods oftransformation and regeneration known in the art also involveintervening single-cell-derived or tissue-proliferation, which comprisetypes of growth patterns referred to as meristem proliferation, whichencompasses de novo meristem formation (Gordon Kamm et al. (2007)Development 134: 3539-3548), multiple shoot formation (Zhong et al.(1992) Planta 187:483-489) and green tissue culture (Cho et al. (1998)Plant Sci 138:229-244; Cho et al. (2004) Plant Cell Rep 22:483-489), allrequiring cytokinin in the medium (e.g., U.S. Pat. No. 8,395,020B2; U.S.Pat. No. 8,581,035B2).

In the disclosed methods, various explants can be used, includingimmature embryos, 1-5 mm zygotic embryos, 3-5 mm embryos, and embryosderived from mature ear-derived seed, leaf bases, leaves from matureplants, leaf tips, immature influorescences, tassel, immature ear, andsilks. In various aspects, the plant-derived explant used fortransformation includes immature embryos, 1-5 mm zygotic embryos, and3.5-5 mm embryos. In an aspect, the embryos used in the disclosedmethods can be derived from mature ear-derived seed, leaf bases, leavesfrom mature plants, leaf tips, immature influorescences, tassel,immature ear, and silks.

Regenerable plant structure is defined as a multicellular structurecapable of forming a fully functional fertile plant, such as, but notlimited to, somatic embryos, embryogenic callus, somatic meristems,and/or organogenic callus.

Somatic embryo is defined as a multicellular structure that progressesthrough developmental stages that are similar to the development of azygotic embryo, including formation of globular and transition-stageembryos, formation of an embryo axis and a scutellum, and accumulationof lipids and starch. Single somatic embryos derived from a zygoticembryo germinate to produce single non-chimeric plants, which mayoriginally derive from a single-cell.

Embryogenic callus is defined as a friable or non-friable mixture ofundifferentiated or partially undifferentiated cells which subtendproliferating primary and secondary somatic embryos capable ofregenerating into mature fertile plants.

Somatic meristem is defined as a multicellular structure that is similarto the apical meristem which is part of a seed-derived embryo,characterized as having an undifferentiated apical dome flanked by leafprimorida and subtended by vascular initials, the apical dome givingrise to an above-ground vegetative plant. Such somatic meristems canform single or fused clusters of meristems.

Organogenic callus is defined as a compact mixture of differentiatedgrowing plant structures, including but not limited to apical meristems,root meristems, leaves and roots.

Germination is the growth of a regenerable structure to form a plantletwhich continues growing to produce a plant.

A transgenic plant is defined as a mature, fertile plant that contains atransgene.

The explant used in the disclosed methods can be derived from a monocot,including, but not limited to, barley, maize, millet, oats, rice, rye,Setaria sp., sorghum, sugarcane, switchgrass, triticale, turfgrass, orwheat. Alternatively, the explant used in the disclosed methods can bederived from a dicot, including, but not limited to, kale, cauliflower,broccoli, mustard plant, cabbage, pea, clover, alfalfa, broad bean,tomato, cassava, soybean, canola, alfalfa, sunflower, safflower,tobacco, Arabidopsis, or cotton.

In a further aspect, the explant used in the disclosed methods can bederived from a plant that is a member of the family Poaceae.Non-limiting examples of suitable plants from which an explant can bederived include grain crops, including, but not limited to, barley,maize (corn), oats, rice, rye, sorghum, wheat, millet, triticale; leafand stem crops, including, but not limited to, bamboo, marram grass,meadow-grass, reeds, ryegrass, sugarcane; lawn grasses, ornamentalgrasses, and other grasses such as switchgrass and turfgrass.

In a further aspect, the explant used in the disclosed methods can bederived from any plant, including higher plants, e.g., classes ofAngiospermae and Gymnospermae. Plants of the subclasses of theDicotylodenae and the Monocotyledonae are suitable. Suitable species maycome from the family Acanthaceae, Alliaceae, Alstroemeriaceae,Amaryllidaceae, Apocynaceae, Arecaceae, Asteraceae, Berberidaceae,Bixaceae, Brassicaceae, Bromeliaceae, Cannabaceae, Caryophyllaceae,Cephalotaxaceae, Chenopodiaceae, Colchicaceae, Cucurbitaceae,Dioscoreaceae, Ephedraceae, Erythroxylaceae, Euphorbiaceae, Fabaceae,Lamiaceae, Linaceae, Lycopodiaceae, Malvaceae, Melanthiaceae, Musaceae,Myrtaceae, Nyssaceae, Papaveraceae, Pinaceae, Plantaginaceae, Poaceae,Rosaceae, Rubiaceae, Salicaceae, Sapindaceae, Solanaceae, Taxaceae,Theaceae, and Vitaceae.

Suitable species from which the explant used in the disclosed methodscan be derived include members of the genus Abelmoschus, Abies, Acer,Agrostis, Allium, Alstroemeria, Ananas, Andrographis, Andropogon,Artemisia, Arundo, Atropa, Berberis, Beta, Bixa, Brassica, Calendula,Camellia, Camptotheca, Cannabis, Capsicum, Carthamus, Catharanthus,Cephalotaxus, Chrysanthemum, Cinchona, Citrullus. Coffea, Colchicum,Coleus, Cucumis, Cucurbita, Cynodon, Datura, Dianthus, Digitalis,Dioscorea, Elaeis, Ephedra, Erianthus, Erythroxylum, Eucalyptus,Festuca, Fragaria, Galanthus, Glycine, Gossypium, Helianthus, Hevea,Hordeum, Hyoscyamus, Jatropha, Lactuca, Linum, Lolium, Lupinus,Lycopersicon, Lycopodium, Manihot, Medicago, Mentha, Miscanthus, Musa,Nicotiana, Oryza, Panicum, Papaver, Parthenium, Pennisetum, Petunia,Phalaris, Phleum, Pinus, Poa, Poinsettia, Populus, Rauwolfia, Ricinus,Rosa, Saccharum, Salix, Sanguinaria, Scopolia, Secale, Solanum, Sorghum,Spartina, Spinacea, Tanacetum, Taxus, Theobroma, Triticosecale,Triticum, Uniola, Veratrum, Vinca, Vitis, and Zea.

In a further aspect, the explant used in the disclosed methods can bederived from a plant that is important or interesting for agriculture,horticulture, biomass for the production of liquid fuel molecules andother chemicals, and/or forestry. Non-limiting examples include, forinstance, Panicum virgatum (switchgrass), Sorghum bicolor (sorghum,sudangrass), Miscanthus giganteus (miscanthus), Saccharum sp. (energycane), Populus balsamifera (poplar), Zea mays (corn), Glycine max(soybean), Brassica napus (canola), Triticum aestivum (wheat), Gossypiumhirsutum (cotton), Oryza sativa (rice), Helianthus annuus (sunflower),Medicago saliva (alfalfa), Beta vulgaris (sugarbeet), Pennisetum glaucum(pearl millet), Panicum spp., Sorghum spp., Miscanthus spp., Saccharumspp., Erianthus spp., Populus spp., Andropogon gerardii (big bluestem),Pennisetum purpureum (elephant grass), Phalaris arundinacea (reed canarygrass), Cynodon dactylon (bermudagrass), Festuca arundinacea (tallfescue), Spartina pectinata (prairie cord-grass), Arundo donax (giantreed), Secale cereale (rye), Salix spp. (willow), Eucalyptus spp.(eucalyptus), Triticosecale spp. (triticum-wheat X rye), Bamboo,Carthamus tinctorius (safflower), Jatropha curcas (jatropha), Ricinuscommunis (castor), Elaeis guineensis (palm), Linum usitatissimum (flax),Brassica juncea. Manihot esculenta (cassava), Lycopersicon esculentum(tomato), Lactuca sativa (lettuce), Musa paradisiaca (banana), Solanumtuberosum (potato), Brassica oleracea (broccoli, cauliflower,brusselsprouts), Camellia sinensis (tea), Fragaria ananassa(strawberry), Theobroma cacao (cocoa), Coffea arabica (coffee), Vitisvinifera (grape), Ananas comosus (pineapple), Capsicum annum (hot &sweet pepper), Allium cepa (onion), Cucumis melo (melon), Cucumissativus (cucumber), Cucurbita maxima (squash), Cucurbita moschata(squash), Spinacea oleracea (spinach), Citrullus lanatus (watermelon),Abelmoschus esculentus (okra), Solanum melongena (eggplant), Papaversomniferum (opium poppy), Papaver orientale. Taxus baccata, Taxusbrevifolia, Artemisia annua, Cannabis sativa, Camptotheca acuminate,Catharanthus roseus, Vinca rosea, Cinchona officinalis, Colchicumautumnale, Veratrum californica, Digitalis lanata, Digitalis purpurea,Dioscorea spp., Andrographis paniculata, Atropa belladonna, Daturastomonium, Berberis spp., Cephalotaxus spp., Ephedra sinica. Ephedraspp., Erythroxylum coca, Galanthus wornorii, Scopolia spp., Lycopodiumserratum (=Huperzia serrata), Lycopodium spp., Rauwolfia serpentina,Rauwolfia spp., Sanguinaria canadensis, Hyoscyamus spp., Calendulaofficinalis, Chrysanthemum parthenium, Coleus forskohlii, Tanacetumparthenium, Parthenium argentatum (guayule), Hevea spp. (rubber), Menthaspicata (mint), Mentha piperita (mint), Bixa orellana, Alstroemeriaspp., Rosa spp. (rose), Dianthus caryophyllus (carnation), Petunia spp.(petunia), Poinsettia pulcherrima (poinsettia), Nicotiana tabacum(tobacco), Lupinus albus (lupin), Uniola paniculata (oats), bentgrass(Agrostis spp.), Populus tremuloides (aspen), Pinus spp. (pine), Abiesspp. (fir), Acer spp. (maple), Hordeum vulgare (barley), Poa pratensis(bluegrass), Lolium spp. (ryegrass), Phleum pratense (timothy), andconifers. Of interest are plants grown for energy production, so calledenergy crops, such as cellulose-based energy crops like Panicum virgatum(switchgrass), Sorghum bicolor (sorghum, sudangrass), Miscanthusgiganteus (miscanthus). Saccharum sp. (energy cane), Populus balsamifera(poplar). Andropogon gerardii (big bluestem), Pennisetum purpureum(elephant grass), Phalaris arundinacea (reed canary grass), Cynodondactylon (bermudagrass), Festuca arundinacea (tall fescue). Spartinapectinata (prairie cord-grass), Medicago saliva (alfalfa), Arundo donax(giant reed), Secale cereale (rye), Salix spp. (willow), Eucalyptus spp.(eucalyptus), Triticosecale spp. (triticum-wheat X rye), and Bamboo; andstarch-based energy crops like Zea mays (corn) and Manihot esculenta(cassava); and sucrose-based energy crops like Saccharum sp. (sugarcane)and Beta vulgaris (sugarbeet); and biodiesel-producing energy crops likeGlycine max (soybean). Brassica napus (canola), Helianthus annuus(sunflower), Carthamus tinctorius (safflower), Jatropha curcas(jatropha), Ricinus communis (castor), Elaeis guineensis (palm), Linumusitatissimum (flax), and Brassica juncea.

As used herein, a “biomass renewable energy source plant” means havingor producing material (either raw or processed) that comprises storedsolar energy that can be converted to electrical energy, liquid fuels,and other useful chemicals. In general terms, such plants comprisededicated energy crops as well as agricultural and woody plants.Examples of biomass renewable energy source plants include: Panicumvirgatum (switchgrass), Sorghum bicolor (sorghum, sudangrass),Miscanthus giganteus (miscanthus), Saccharum sp. (energy cane), Populusbalsamifera (poplar). Andropogon gerardii (big bluestem), Pennisetumpurpureum (elephant grass), Phalaris arundinacea (reed canary grass),Cynodon dactylon (bermudagrass), Festuca arundinacea (tall fescue),Spartina pectinata (prairie cord-grass), Medicago sativa (alfalfa),Arundo donax (giant reed), Secale cereale (rye), Salix spp. (willow),Eucalyptus spp. (eucalyptus), Triticosecale spp. (triticum-wheat X rye),Bamboo, Zea mays (corn), Manihot esculenta (cassava), Saccharum sp.(sugarcane), Beta vulgaris (sugarbeet), Glycine max (soybean), Brassicanapus (canola), Helianthus annuus (sunflower), Carthamus tinctorius(safflower), Jatropha curcas (jatropha), Ricinus communis (castor),Elaeis guineensis (palm), Linum usitatissimum (flax), and Brassicajuncea.

In an aspect, the expression construct comprises both a nucleotidesequence encoding a WUS/WOX homeobox polypeptide and a nucleotidesequence encoding a polypeptide comprising two AP2-DNA binding domains.In various aspects, expression of a nucleotide sequence encoding aWUS/WOX homeobox polypeptide and a nucleotide sequence encoding apolypeptide comprising two AP2 binding domains occurs for less than 1day, less than 2 days, less than 5 days, less than 7 days, or less than14 days after initiation of transformation.

In an aspect, the expression construct comprises a nucleotide sequenceencoding a WUS/WOX homeobox polypeptide. In various aspects, expressionof a nucleotide sequence encoding a WUS/WOX homeobox polypeptide occursfor less than 1 day, less than 2 days, less than 5 days, less than 7days, or less than 14 days after initiation of transformation.

In an aspect, the expression construct comprises a nucleotide sequenceencoding a polypeptide comprising two AP2-DNA binding domains. Invarious aspects, expression of a nucleotide sequence encoding apolypeptide comprising two AP2 binding domains occurs for less than 1day, less than 2 days, less than 5 days, less than 7 days, or less than14 days after initiation of transformation.

In an aspect, the nucleotide sequence encoding the WUS/WOX homeoboxpolypeptide; the nucleotide sequence encoding a polypeptide comprisingtwo AP2-DNA binding domains; or both nucleotide sequences can betargeted for excision by a site-specific recombinase. Thus, theexpression of the nucleotide sequence encoding the WUS/WOX homeoboxpolypeptide; the nucleotide sequence encoding a polypeptide comprisingtwo AP2-DNA binding domains; or both nucleotide sequences can becontrolled by excision at a desired time post-transformation. It isunderstood that when a site-specific recombinase is used to control theexpression of the nucleotide sequence encoding the WUS/WOX homeoboxpolypeptide; the nucleotide sequence encoding a polypeptide comprisingtwo AP2-DNA binding domains; or both nucleotide sequences in theexpression construct, the expression construct comprises appropriatesite-specific excision sites flanking the polynucleotide sequences to beexcised, e.g., Cre lox sites if Cre recombinase is utilized. It is notnecessary that the site-specific recombinase be co-located on theexpression construct comprising the nucleotide sequence encoding theWUS/WOX homeobox polypeptide; the nucleotide sequence encoding apolypeptide comprising two AP2-DNA binding domains; or both nucleotidesequences. However, in an aspect, the expression construct furthercomprises a nucleotide sequence encoding a site-specific recombinase.

The site-specific recombinase used to control expression of thenucleotide sequence encoding the WUS/WOX homeobox polypeptide; thenucleotide sequence encoding a polypeptide comprising two AP2-DNAbinding domains; or both polynucleotide sequences, can be chosen from avariety of suitable site-specific recombinases. For examples, in variousaspects, the site-specific recombinase is FLP, Cre. SSV1, lambda Int,phi C31 Int, HK022, R, Gin, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1,or U153. The site-specific recombinase can be a destabilized fusionpolypeptide. The destabilized fusion polypeptide can be TETR(G17A)˜CREor ESR(G17A)˜CRE.

In an aspect, the nucleotide sequence encoding a site-specificrecombinase is operably linked to a constitutive promoter, an induciblepromoter, or a developmentally-regulated promoter. Suitabledevelopmentally regulated promoters include, GLB1, OLE, and LipidTransfer Protein2 (LTP2) (Kalla et al., 1994. Plant J. 6:849-860 andU.S. Pat. No. 5,525,716). Suitable inducible promoters includeenvironmentally inducible heat shock promoters including HSP17.7, HSP26,or HSP18A. Alternatively, the inducible promoter operably linked to thesite-specific recombinase can be a chemically inducible promoter, forexample the XVE promoter.

In an aspect, the chemically inducible promoter operably linked to thesite-specific recombinase is XVE. The chemically-inducible promoter canbe repressed by the tetraycline repressor (TETR), the ethametsulfuronrepressor (ESR), or the chlorsulfuron repressor (CR), and de-repressionoccurs upon addition of tetracycline-related or sulfonylurea ligands.The repressor can be TETR and the tetracycline-related ligand isdoxycycline or anhydrotetracycline. (Gatz, C., Frohberg, C. andWendenburg, R. (1992) Stringent repression and homogeneous de-repressionby tetracycline of a modified CaMV 35S promoter in intact transgenictobacco plants, Plant J. 2, 397-404). Alternatively, the repressor canbe ESR and the sulfonylurea ligand is ethametsulfuron, chlorsulfuron,metsulfuron-methyl, sulfometuron methyl, chlorimuron ethyl,nicosulfuron, primisulfuron, tribenuron, sulfosulfuron,trifloxysulfuron, foramsulfuron, iodosulfuron, prosulfuron,thifensulfuron, rimsulfuron, mesosulfuron, or halosulfuron(US20110287936 incorporated herein by reference in its entirety).

In an aspect, when the expression construct comprises site-specificrecombinase excision sites, the nucleotide sequence encoding the WUS/WOXhomeobox polypeptide; the nucleotide sequence encoding a polypeptidecomprising two AP2-DNA binding domains; or both nucleotide sequences canbe operably linked to an auxin inducible promoter, a developmentallyregulated promoter, or a constitutive promoter. Exemplary constitutivepromoters useful in this context include UBI, LLDAV, EVCV, DMMV, BSV(AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO (ALT1), USB1ZMPRO, ZM-GOS2 PRO, ZM-HIB PRO (1.2 KB), IN2-2, NOS, the -135 version ofthe 35S PRO (or longer versions of the 35S promoter, and ZM-ADF PRO(ALT2). Exemplary auxin inducible promoters useful in this contextinclude AXIG1 and DR5. Exemplary developmentally regulated promotersuseful in this context include PLTP, PLTP1, PLTP2, PLTP3, LGL, LEA-14A,and LEA-D34.

The appropriate duration for expression of the nucleotide sequenceencoding a WUS/WOX homeobox polypeptide; the nucleotide sequenceencoding a polypeptide comprising two AP2-DNA binding domains; or bothnucleotide sequences can be achieved by use of adevelopmentally-regulated promoter, e.g., a promoter such as aPhospholipid Transfer Protein (PLTP). PLTP promoters include PLTP,PLTP1, PLTP2, and PLTP3 as further described below. In a particularaspect, the PLTP promoter comprises any one of SEQ ID NO: 1-2, and55-61. The PLTP promoter used can be a Zea mays or Sorghum bicolor PLTPpromoter such as that of SEQ ID NO: 1 or 2 (U.S. Provisional Appl. No.62/271,230 herein incorporated by reference in its entirety). Otherappropriate developmentally-regulated promoters that can be used withthe methods of the present disclosure include the promoters derived fromgenes encoding the fructose-1,6-bisphosphatase protein, NAD(P)-bindingRossmann-Fold protein, adipocyte plasma membrane-associated protein-likeprotein, Rieske [2Fe-2S] iron-sulfur domain protein, chlororespiratoryreduction 6 protein, D-glycerate 3-kinase, chloroplastic-like protein,chlorophyll a-b binding protein 7, chloroplastic-like protein,ultraviolet-B-repressible protein, Soul heme-binding family protein,Photosystem I reaction center subunit psi-N protein, and short-chaindehydrogenase/reductase protein. The promoters can be derived from thegenes encoding the foregoing proteins in monocot plants, such as maize,rice, or sorghum. Suitable developmentally-regulated promoters useful inthe methods of the present disclosure include those shown in SEQ ID NOs:1-2, 28-40, 55-61, 81-83 and 86-88 and 106.

In an aspect, the nucleotide sequence encoding the WUS/WOX homeoboxpolypeptide; the nucleotide sequence encoding a polypeptide comprisingtwo AP2-DNA binding domains; or both polynucleotide sequences can beoperably linked to an auxin-inducible promoter. In various aspects, theauxin-inducible promoter can be AXIG1. In particular, theauxin-inducible promoter can be SEQ ID NO:39. The promoter can alsocomprise one or more auxin-response elements. In a further aspect, thepromoter can comprise one or more DR5 motifs. In particular, thepromoter can be SEQ ID NO:40.

In an aspect, the promoter is a weak constitutive promoter modified forrepression and de-repression. In a further aspect, the weak constitutivepromoter modified for repression and de-repression comprises one or moreoperator sequences in the promoter that have been positioned near oroverlapping the TATA box and/or the transcription start site. Forexample, suitable promoters of this type include, but are not limitedto, the NOS, IN2-2, the -135 version of 35S, CC-UB11-PRO and ZM-ADF4-PROpromoters.

In an aspect, the nucleotide sequence encoding the WUS/WOX homeoboxpolypeptide; the nucleotide sequence encoding a polypeptide comprisingtwo AP2-DNA binding domains; or both nucleotide sequences can beoperably linked to a developmentally-regulated promoter. Suitabledevelopmentally-regulated promoters include promoters derived from theplant genes for Fructose-1,6-bisphosphatase, NAD(P)-bindingRossmann-Fold Protein, Adipocyte plasma membrane-associated protein-likeprotein, Rieske [2Fe-2S] iron-sulphur domain protein, Chlororespiratoryreduction 6 protein, D-glycerate 3-kinase, chloroplastic-like protein,Chlorophyll a-b binding protein 7, chloroplastic-like protein,Ultraviolet-B-repressible protein, Soul heme-binding family protein,Photosystem I reaction center subunit psi-N protein, and Short-chaindehydrogenase/reductase. In various aspects, these promoters are derivedfrom the corresponding genes in monocots such as maize, rice, and thelike. Alternatively, these promoters can be derived from thecorresponding genes in dicots. In particular aspects, thedevelopmentally-regulated promoters comprise any one of SEQ ID NOS: 1-2,28-40, 55-61, 81-83 and 86-88 and 106.

In an aspect, the nucleotide sequence encoding the WUS/WOX homeoboxpolypeptide; the nucleotide sequence encoding a polypeptide comprisingtwo AP2-DNA binding domains; or both nucleotide sequences can beoperably linked to a de-repressible promoter. Useful de-repressiblepromoters include 35S:Top3, NOS::Top, NOS:Top2, UBI:Top3, BSV:Top3,AXIG1:Top, IN2-2:Top and DR5:Top (where Top is the abbreviation of the18-base operator sequence to which either TETR, ESR or CR bind torepress expression).

In an aspect, the present disclosure provides transformation methodsthat allow positive growth selection. One skilled in the art canappreciate that conventional plant transformation methods have reliedpredominantly on negative selection schemes, in which an antibiotic orherbicide (a negative selective agent) is used to inhibit or killnon-transformed cells or tissues, and the transgenic cells or tissuescontinue to grow due to expression of a resistance gene. In contrast,the methods of the present disclosure can be used with no application ofa negative selective agent. Thus, although wild-type cells can growunhindered, by comparison cells containing the disclosed WUS2 and ODP2expression cassettes can be readily identified due to their acceleratedgrowth rate relative to the surrounding wild-type tissue. In addition tosimply observing faster growth, the methods of the present disclosureprovide transgenic cells containing the WUS2 and ODP2 expressioncassettes that exhibit more rapid morphogenesis, manifested asregenerable plant structure formation, relative to non-transformedcells. Accordingly, such differential growth and morphogenic developmentcan be used to easily distinguish transgenic regenerable plantstructures from the surrounding non-transformed tissue, a process whichis termed herein as “positive growth selection.”

In various aspects, the methods of the present disclosure can be carriedout without using a rooting medium. Alternatively, in an aspect, themethods of the present disclosure can be carried out using a rootingmedium. As used herein, “rooting medium” or “selective rooting medium”refers to a tissue culture medium comprising basal salts, carbonsources, vitamins, minerals and plant phytohormones. In an aspect, theplant phytohormones can be provided at varying concentrations or ratios,wherein root tissues develop and proliferate from cells placed upon theselective rooting medium. In an aspect, the selective rooting mediumcontains glufosinate, imazapyr, ethametsulfuron, mannose or otherselective agents. In an aspect, the selective rooting medium containsauxin and cytokinin, cytokinin alone, or no plant phytohormones.

In an aspect, the methods of the present disclosure are carried outwithout using a cytokinin during regenerable plant structure formationand/or during germination. In a further aspect, the methods of thepresent disclosure can be carried out without using a cytokinin betweentransformation and at least about 7 days after initiatingtransformation. In a further aspect, the methods of the presentdisclosure can be carried out without using a cytokinin betweentransformation and at least about 14 days after initiatingtransformation.

In an aspect, the nucleotide sequence encoding the WUS/WOX homeoboxpolypeptide; the nucleotide sequence encoding a polypeptide comprisingtwo AP2-DNA binding domains; or each nucleotide sequence is operablylinked to a PLTP promoter. PLTP promoters include PLTP, PLTP1, PLTP2,and PLTP3. The PLTP promoter can be derived from a monocot, including,but not limited to, maize, barley, oats, rye, sugarcane, millet,sorghum, wheat. Setaria sp., or rice. In a further aspect, the PLTPpromoter is a maize, rice, or Setaria sp. PLTP promoter. In a particularaspect, the PLTP promoter comprises SEQ ID NO: 1-2 and 55-61. In aparticular aspect, the PLTP promoter is a maize or rice PLTP promoter.In a further aspect, the PLTP promoter is a maize PLTP promoter. In afurther aspect, the PLTP promoter is a rice PLTP promoter. In an aspect,the PLTP promoter comprises SEQ ID NO: 1 or 2. The PLTP promoter canalso be derived from a dicot, including, but not limited to, kale,cauliflower, broccoli, mustard plant, cabbage, pea, clover, alfalfa,broad bean, tomato, cassava, soybean, canola, alfalfa, sunflower,safflower, tobacco, Arabidopsis, or cotton. Other developmentallyregulated promoters useful in this context include LGL, LEA-14A, andLEA-D34. In a particular aspect, other promoters useful in the presentmethods include any one of SEQ ID NO: 28-40, 81-83, 86-88, and 106.

In an aspect, the present disclosure comprises methods for producing atransgenic plant, comprising (a) transforming a cell of an explant withan expression construct comprising: (i) a nucleotide sequence encoding aWUS/WOX homeobox polypeptide; (ii) a nucleotide sequence encoding apolypeptide comprising two AP2-DNA binding domains; or (iii) acombination thereof, and (b) allowing expression of a polypeptide of (a)in each transformed cell to form a regenerable plant structure in theabsence of exogenous cytokinin, wherein no callus is formed; and (c)germinating the regenerable plant structure to form the transgenicplant; wherein the WUS/WOX homeobox polypeptide comprises the amino acidsequence of any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16; or whereinthe WUS/WOX homeobox polypeptide is encoded by the nucleotide sequenceof any one of SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15; and wherein thepolypeptide comprising two AP2-DNA binding domains comprises the aminoacid sequence of any one of SEQ ID NO: 18, 20, 63, 65, or 67; or whereinthe polypeptide comprising two AP2-DNA binding domains is encoded by thenucleotide sequence of any one of SEQ ID NO: 17, 19, 21, 62, 64, 66, or68.

The regenerable plant structure is produced within about 0-7 days orabout 0-14 days of transformation. In an aspect, the germinatingcomprises transferring the regenerable plant structure to a maturationmedium comprising an exogenous cytokinin and forming the transgenicplant. In an aspect, the nucleotide sequence encoding a WUS/WOX homeoboxpolypeptide is capable of stimulating formation of a regenerable plantstructure. In an aspect, the nucleotide sequence encoding a polypeptidecomprising two AP2-DNA binding domains is capable of stimulatingformation of a regenerable plant structure.

In an aspect, the present disclosure comprises methods for producing atransgenic plant, comprising (a) transforming a cell of an explant withan expression construct comprising: (i) a nucleotide sequence encoding aWUS2 polypeptide; (ii) a nucleotide sequence encoding a ODP2polypeptide; or (iii) a combination thereof; and (b) allowing expressionof a polypeptide of (a) in each transformed cell to form a regenerableplant structure in the absence of exogenous cytokinin, wherein no callusis formed; and (c) germinating the regenerable plant structure to formthe transgenic plant; wherein the WUS2 polypeptide comprises the aminoacid sequence of SEQ ID NO: 4; or wherein the WUS2 polypeptide isencoded by the nucleotide sequence of SEQ ID NO: 3; and wherein the ODP2polypeptide comprises the amino acid sequence of SEQ ID NO: 18, 63, 65,or 67; or wherein the polypeptide comprising two AP2-DNA binding domainsis encoded by the nucleotide sequence of any one of SEQ ID NO: 17, 21,62, 64, 66, or 68.

In an aspect, the present disclosure comprises methods for producing atransgenic plant, comprising (a) transforming a cell of an explant withan expression construct comprising a nucleotide sequence encoding aWUS/WOX homeobox polypeptide; and (b) allowing expression of thepolypeptide of (a) in each transformed cell to form a regenerable plantstructure in the absence of exogenous cytokinin, wherein no callus isformed; and (c) germinating the regenerable plant structure to form thetransgenic plant.

In an aspect, the present disclosure comprises methods for producing atransgenic plant, comprising (a) transforming one or more cells of anexplant with an expression construct comprising a nucleotide sequenceencoding a polypeptide comprising two AP2-DNA binding domains; and (b)allowing expression of the polypeptide of (a) in each transformed cellto form a regenerable plant structure in the absence of exogenouscytokinin, wherein no callus is formed; and (c) germinating theregenerable plant structure to form the transgenic plant.

In an aspect, the present disclosure comprises methods for producing atransformed plant, comprising (a) transforming one or more cells of anexplant with an expression construct comprising (i) a nucleotidesequence encoding a WUS/WOX homeobox polypeptide; and (ii) a nucleotidesequence encoding a polypeptide comprising two AP2-DNA binding domains;and (b) allowing expression of the polypeptides of (a) in eachtransformed cell to form a regenerable plant structure in the absence ofexogenous cytokinin, wherein no callus is formed; and (c) germinatingthe regenerable plant structure to form the transgenic plant.

In an aspect, the present disclosure comprises methods for producing atransformed plant, comprising (a) transforming a cell of an explant withan expression construct comprising: (i) a nucleotide sequence encoding aWUS/WOX homeobox polypeptide; (ii) a nucleotide sequence encoding apolypeptide comprising two AP2-DNA binding domains; or (iii) acombination of (i) and (ii); (b) allowing expression of the polypeptideof (a) in each transformed cell to form a regenerable plant structure inthe absence of exogenous cytokinin, wherein no callus is formed; and (c)germinating the regenerable plant structure to form the transgenic plantin about 14 days to about 60 days.

The term “plant” refers to whole plants, plant organs, plant tissues,seeds, plant cells, seeds and progeny of the same. Plant cells include,without limitation, cells from seeds, suspension cultures, embryos,meristematic regions, callus tissue, leaves, roots, shoots,gametophytes, sporophytes, pollen and microspores.

Plant parts include differentiated and undifferentiated tissuesincluding, but not limited to the following: roots, stems, shoots,leaves, pollen, seeds, tumor tissue and various forms of cells andculture (e.g., single cells, protoplasts, embryos and callus tissue).The plant tissue may be in a plant or in a plant organ, tissue or cellculture.

The present disclosure also includes plants obtained by any of thedisclosed methods or compositions herein.

In an aspect, the present disclosure comprises methods for producing atransgenic plant, comprising (a) transforming a cell of an explant withan expression construct comprising: (i) a nucleotide sequence encoding aWUS/WOX homeobox polypeptide; (ii) a nucleotide sequence encoding apolypeptide comprising two AP2-DNA binding domains; or (iii) acombination of (i) and (ii); (b) allowing expression of the polypeptideof (a) in each transformed cell to form a regenerable plant structure inthe absence of exogenous cytokinin, wherein no callus is formed; and (c)germinating the regenerable plant structure to form the transgenic plantin about 14 days to about 60 days.

The present disclosure also includes seeds from a plant obtained by anyof the disclosed methods or compositions herein.

In an aspect, the present disclosure comprises kits comprising: (a) anexpression construct comprising (i) a nucleotide sequence encoding aWUS/WOX homeobox polypeptide; (ii) a nucleotide sequence encoding apolypeptide comprising two AP2-DNA binding domains; or (iii) acombination of (i) and (ii); and (b) instructions for obtaining aregenerable plant structure in the absence of exogenous cytokinin,wherein no callus is formed; and (c) instructions for germinating theregenerable plant structure to form the transgenic plant.

In an aspect, the present disclosure comprises kits comprising (a) anexpression construct comprising a nucleotide sequence encoding a WUS/WOXhomeobox polypeptide; and (b) instructions for obtaining a regenerableplant structure in the absence of exogenous cytokinin, wherein no callusis formed; and (c) instructions for germinating the regenerable plantstructure to form the transgenic plant.

In an aspect, the present disclosure comprises kits comprising (a) anexpression construct comprising a nucleotide sequence encoding apolypeptide comprising two AP2-DNA binding domains; and (b) instructionsfor obtaining a regenerable plant structure in the absence of exogenouscytokinin, wherein no callus is formed; and (c) instructions forgerminating the regenerable plant structure to form the transgenicplant.

The disclosed kits can further comprise instructions for germinating theregenerable plant structure to form the transgenic plant within about 14days to about 60 days after transformation of the explant.

II. AP2-DNA Binding Domain Proteins (ODP2/BBM)

The methods of the present disclosure comprise polynucleotide sequencesand amino acid sequences of Ovule Development Protein 2 (ODP2)polypeptides, and related polypeptides, e.g., Baby boom (BBM) proteinfamily proteins. In an aspect, the polypeptide comprising the twoAP2-DNA binding domains is an ODP2, BBM2, BMN2, or BMN3 polypeptide. TheODP2 polypeptides of the disclosure contain two predicted APETALA2 (AP2)domains and are members of the AP2 protein family (PFAM AccessionPF00847). The AP2 domains of the maize ODP2 polypeptide are located fromabout amino acids S273 to N343 and from about S375 to R437 of SEQ IDNO:2). The AP2 family of putative transcription factors has been shownto regulate a wide range of developmental processes, and the familymembers are characterized by the presence of an AP2 DNA binding domain,his conserved core is predicted to form an amphipathic alpha helix thatbinds DNA. The AP2 domain was first identified in APETALA2, anArabidopsis protein that regulates meristem identity, floral organspecification, seed coat development, and floral homeotic geneexpression. The AP2 domain has now been found in a variety of proteins.

The ODP2 polypeptides of the disclosure share homology with severalpolypeptides within the AP2 family, e.g., see FIG. 1 of U.S. Pat. No.8,420,893, which is incorporated herein by reference in its entirety,provides an alignment of the maize and rice ODP2 polypeptides with eightother proteins having two AP2 domains. A consensus sequence of allproteins appearing in the alignment of U.S. Pat. No. 8,420,893 is alsoprovided in FIG. 1 therein.

The polypeptide comprising the two AP2-DNA binding domains can bederived from a monocot. In various aspects, the polypeptide comprisingthe two AP2-DNA binding domains is derived from barley, maize, millet,oats, rice, rye, Setaria sp., sorghum, sugarcane, switchgrass,triticale, turfgrass, or wheat. In an aspect, the polypeptide comprisingthe two AP2-DNA binding domains is derived from maize, barley, oats,rye, sugarcane, millet, sorghum, wheat, Setaria sp., or rice. In afurther aspect, the monocot is maize, sorghum, rice, or Setaria sp. In astill further aspect, the monocot is maize or rice. In a further aspect,the monocot is maize. In a further aspect, the monocot is rice.

The polypeptide comprising the two AP2-DNA binding domains can bederived from a dicot. The polypeptide comprising the two AP2-DNA bindingdomains can be derived from kale, cauliflower, broccoli, mustard plant,cabbage, pea, clover, alfalfa, broad bean, tomato, cassava, soybean,canola, alfalfa, sunflower, safflower, tobacco, Arabidopsis, or cotton.

The polypeptide comprising the two AP2-DNA binding domains can bederived from a plant that is a member of the family Poaceae.Non-limiting examples of suitable plants from which the two AP2-DNAbinding domains can be derived include grain crops, including, but notlimited to, barley, maize (corn), oats, rice, rye, sorghum, wheat,millet, triticale; leaf and stem crops, including, but not limited to,bamboo, marram grass, meadow-grass, reeds, ryegrass, sugarcane; lawngrasses, ornamental grasses, and other grasses such as switchgrass andturfgrass.

In a further aspect, the polypeptide comprising the two AP2-DNA bindingdomains used in the disclosed methods can be derived from any plant,including higher plants, e.g., classes of Angiospermae and Gymnospermae.Plants of the subclasses of the Dicotylodenae and the Monocotyledonaeare suitable. Suitable species may come from the family Acanthaceae,Alliaceae, Alstroemeriaceae, Amaryllidaceae, Apocynaceae, Arecaceae,Asteraceae, Berberidaceae, Bixaceae, Brassicaceae, Bromeliaceae,Cannabaceae, Caryophyllaceae, Cephalotaxaceae, Chenopodiaceae,Colchicaceae, Cucurbitaceae, Dioscoreaceae, Ephedraceae,Erythroxylaceae, Euphorbiaceae, Fabaceae, Lamiaceae, Linaceae,Lycopodiaceae, Malvaceae, Melanthiaceae, Musaceae, Myrtaceae, Nyssaceae,Papaveraceae, Pinaceae, Plantaginaceae, Poaceae, Rosaceae, Rubiaceae,Salicaceae, Sapindaceae. Solanaceae, Taxaceae, Theaceae, and Vitaceae.

Suitable species from which the polypeptide comprising the two AP2-DNAbinding domains can be derived include members of the genus Abelmoschus,Abies, Acer, Agrostis, Allium, Alstroemeria, Ananas, Andrographis,Andropogon, Artemisia, Arundo, Atropa, Berberis, Beta, Bixa, Brassica,Calendula, Camellia, Camptotheca, Cannabis, Capsicum, Carthamus,Catharanthus, Cephalotaxus, Chrysanthemum, Cinchona, Citrullus, Coffea,Colchicum, Coleus, Cucumis, Cucurbita, Cynodon, Datura, Dianthus,Digitalis, Dioscorea, Elaeis, Ephedra, Erianthus, Erythroxylum,Eucalyptus, Festuca, Fragaria, Galanthus, Glycine, Gossypium,Helianthus, Hevea, Hordeum, Hyoscyamus, Jatropha, Lactuca, Linum,Lolium, Lupinus, Lycopersicon, Lycopodium, Manihot, Medicago, Mentha,Miscanthus, Musa, Nicotiana, Oryza, Panicum, Papaver, Parthenium,Pennisetum, Petunia, Phalaris, Phleum, Pinus, Poa, Poinsettia, Populus,Rauwolfia, Ricinus, Rosa, Saccharum, Salix, Sanguinaria, Scopolia,Secale, Solanum, Sorghum, Spartina, Spinacea, Tanacetum, Taxus,Theobroma, Triticosecale, Triticum, Uniola, Veratrum, Vinca, Vitis, andZea.

In a further aspect, the polypeptide comprising the two AP2-DNA bindingdomains can be derived from a plant that is important or interesting foragriculture, horticulture, biomass for the production of liquid fuelmolecules and other chemicals, and/or forestry. Non-limiting examplesinclude, for instance, Panicum virgatum (switchgrass), Sorghum bicolor(sorghum, sudangrass), Miscanthus giganteus (miscanthus), Saccharum sp.(energy cane), Populus balsamifera (poplar), Zea mays (corn), Glycinemax (soybean), Brassica napus (canola), Triticum aestivum (wheat),Gossypium hirsutum (cotton), Oryza sativa (rice), Helianthus annuus(sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugarbeet),Pennisetum glaucum (pearl millet), Panicum spp., Sorghum spp.,Miscanthus spp., Saccharum spp., Erianthus spp., Populus spp.,Andropogon gerardii (big bluestem), Pennisetum purpureum (elephantgrass), Phalaris arundinacea (reed canary grass), Cynodon dactylon(bermudagrass), Festuca arundinacea (tall fescue), Spartina pectinata(prairie cord-grass), Arundo donax (giant reed), Secale cereale (rye),Salix spp. (willow), Eucalyptus spp. (eucalyptus), Triticosecale spp.(triticum-wheat X rye), Bamboo, Carthamus tinctorius (safflower),Jatropha curcas (jatropha), Ricinus communis (castor), Elaeis guineensis(palm), Linum usitatissimum (flax), Brassica juncea. Manihot esculenta(cassava), Lycopersicon esculentum (tomato), Lactuca sativa (lettuce),Musa paradisiaca (banana), Solanum tuberosum (potato), Brassica oleracea(broccoli, cauliflower, brusselsprouts), Camellia sinensis (tea),Fragaria ananassa (strawberry), Theobroma cacao (cocoa), Coffea arabica(coffee), Vitis vinifera (grape), Ananas comosus (pineapple), Capsicumannum (hot & sweet pepper), Allium cepa (onion), Cucumis melo (melon),Cucumis sativus (cucumber), Cucurbita maxima (squash), Cucurbitamoschata (squash), Spinacea oleracea (spinach), Citrullus lanatus(watermelon), Abelmoschus esculentus (okra), Solanum melongena(eggplant), Papaver somniferum (opium poppy), Papaver orientale. Taxusbaccata, Taxus brevifolia, Artemisia annua, Cannabis sativa, Camptothecaacuminate, Catharanthus roseus, Vinca rosea, Cinchona officinalis,Colchicum autumnale, Veratrum californica, Digitalis lanata, Digitalispurpurea, Dioscorea spp., Andrographis paniculata, Atropa belladonna,Datura stomonium, Berberis spp., Cephalotaxus spp., Ephedra sinica,Ephedra spp., Erythroxylum coca, Galanthus wornorii, Scopolia spp.,Lycopodium serratum (=Huperzia serrata), Lycopodium spp., Rauwolfiaserpentina, Rauwolfia spp., Sanguinaria canadensis, Hyoscyamus spp.,Calendula officinalis, Chrysanthemum parthenium. Coleus forskohlii,Tanacetum parthenium, Parthenium argentatum (guayule), Hevea spp.(rubber), Mentha spicata (mint), Mentha piperita (mint), Bixa orellana,Alstroemeria spp., Rosa spp. (rose). Dianthus caryophyllus (carnation),Petunia spp. (petunia), Poinsettia pulcherrima (poinsettia), Nicotianatabacum (tobacco), Lupinus albus (lupin), Uniola paniculata (oats),bentgrass (Agrostis spp.), Populus tremuloides (aspen), Pinus spp.(pine), Abies spp. (fir), Acer spp. (maple), Hordeum vulgare (barley).Poa pratensis (bluegrass), Lolium spp. (ryegrass), Phleum pratense(timothy), and conifers. Of interest are plants grown for energyproduction, so called energy crops, such as cellulose-based energy cropslike Panicum virgatum (switchgrass), Sorghum bicolor (sorghum,sudangrass), Miscanthus giganteus (miscanthus), Saccharum sp. (energycane), Populus balsamifera (poplar), Andropogon gerardii (big bluestem),Pennisetum purpureum (elephant grass), Phalaris arundinacea (reed canarygrass), Cynodon dactylon (bermudagrass), Festuca arundinacea (tallfescue), Spartina pectinata (prairie cord-grass), Medicago sativa(alfalfa), Arundo donax (giant reed), Secale cereale (rye), Salix spp.(willow), Eucalyptus spp. (eucalyptus), Triticosecale spp.(triticum-wheat X rye), and Bamboo; and starch-based energy crops likeZea mays (corn) and Manihot esculenta (cassava); and sucrose-basedenergy crops like Saccharum sp. (sugarcane) and Beta vulgaris(sugarbeet); and biodiesel-producing energy crops like Glycine max(soybean), Brassica napus (canola), Helianthus annuus (sunflower),Carthamus tinctorius (safflower), Jatropha curcas (jatropha), Ricinuscommunis (castor), Elaeis guineensis (palm), Linum usitatissimum (flax),and Brassica juncea.

The polypeptide comprising the two AP2-DNA binding domains can bederived from a biomass renewable energy source plant. Examples ofbiomass renewable energy source plants include those described hereinabove.

In particular, the present disclosure provides for isolated nucleic acidmolecules comprising nucleotide sequences encoding the amino acidsequences shown in SEQ ID NO: 18, 20, 63, 65, and 67. Further providedare polypeptides having an amino acid sequence encoded by a nucleic acidmolecule (SEQ ID NO: 17, 19, 21, 62, 64, 66, and 68) described herein,and fragments and variants thereof.

In an aspect, the polypeptide comprising the two AP2-DNA binding domainsis an ODP2 polypeptide. In a further aspect, the ODP2 polypeptide isderived from a monocot polypeptide. In particular aspects, the ODP2polypeptide is derived from maize, barley, oats, rye, sugarcane, millet,sorghum, wheat, Setaria sp., or rice. In an aspect, the ODP2 polypeptideis derived from maize, rice, or Setaria sp. In an aspect, the ODP2polypeptide is derived from maize or rice. In a particular aspect, theODP2 polypeptide is derived from maize. In a particular aspect, the ODP2polypeptide is derived from rice. In a further aspect, the ODP2polypeptide is derived from a dicot polypeptide. In particular aspects,the ODP2 polypeptide is derived from kale, cauliflower, broccoli,mustard plant, cabbage, pea, clover, alfalfa, broad bean, tomato,cassava, soybean, canola, alfalfa, sunflower, safflower, tobacco,Arabidopsis, or cotton. The ODP2 polypeptide expressed by the expressionconstruct of the disclosed methods and compositions can be thepolypeptide comprising SEQ ID NO: 18, 63, 65, or 67. The ODP2polypeptide can be encoded by the polynucleotide of SEQ ID NO: 17, 21,62, 64, 66, or 68.

In an aspect, the polypeptide comprising the two AP2-DNA binding domainsis a BBM2 polypeptide. The BBM2 polypeptide can be a monocotpolypeptide, including, but not limited to such monocots as maize,barley, oats, rye, sugarcane, millet, sorghum, wheat, Setaria sp., orrice. In particular aspects, the monocot is maize, sugarcane, rice, orSetaria sp. In a further particular aspect, the monocot is maize orrice. In a still further particular aspect, the monocot is maize.Alternatively, the BBM2 polypeptide can be a dicot polypeptide,including, but not limited to, wherein the dicot is kale, cauliflower,broccoli, mustard plant, cabbage, pea, clover, alfalfa, broad bean,tomato, cassava, soybean, canola, alfalfa, sunflower, safflower,tobacco, Arabidopsis, or cotton. In one aspect, the BBM2 polypeptidecomprises the amino acid sequence of SEQ ID NO:20. In a further aspect,the BBM2 polypeptide is encoded by a nucleotide sequence comprising thesequence of SEQ ID NO:19.

The present disclosure encompasses isolated or substantially purifiednucleic acid or protein ODP2/BBM compositions. An “isolated” or“purified” nucleic acid molecule or protein, or biologically activeportion thereof, is substantially or essentially free from componentsthat normally accompany or interact with the nucleic acid molecule orprotein as found in its naturally occurring environment. Thus, anisolated or purified nucleic acid molecule or protein is substantiallyfree of other cellular material or culture medium when produced byrecombinant techniques, or substantially free of chemical precursors orother chemicals when chemically synthesized. Optimally, an “isolated”nucleic acid is free of sequences (optimally protein encoding sequences)that naturally flank the nucleic acid (i.e., sequences located at the 5′and 3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For example, in various aspects, theisolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturallyflank the nucleic acid molecule in genomic DNA of the cell from whichthe nucleic acid is derived. A protein that is substantially free ofcellular material includes preparations of protein having less thanabout 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein.When the protein of the disclosure or biologically active portionthereof is recombinantly produced, optimally culture medium representsless than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemicalprecursors or non-protein-of-interest chemicals.

Fragments and variants of the disclosed nucleotide sequences andproteins encoded thereby are also encompassed by the present disclosure.By “fragment” is intended a portion of the nucleotide sequence or aportion of the amino acid sequence and hence protein encoded thereby.Fragments of a nucleotide sequence may encode protein fragments thatretain the biological activity of the native protein and hence have ODP2activity. Alternatively, fragments of a nucleotide sequence useful ashybridization probes generally do not encode fragment proteins retainingbiological activity. Thus, fragments of a nucleotide sequence may rangefrom at least about 20 nucleotides, about 50 nucleotides, about 100nucleotides, and up to the full-length nucleotide sequence encoding theproteins of the disclosure.

By “ODP2 activity” or “Ovule Development Protein 2 activity” is intendedthe ODP2 polypeptide has at least one of the following exemplaryactivities: increases the regenerative capability of a plant cell;renders the plant cell embryogenic; increases the transformationefficiencies of a plant cell; alters the oil content of a plant cell;binds DNA; increases abiotic stress tolerance; increases or maintainsyield under abiotic stress; increases asexual embryo formation; altersstarch content; alters embryo size or activates transcription. Methodsto assay for such activity are known in the art and are described morefully below.

A fragment of an ODP2/BBM nucleotide sequence that encodes abiologically active portion of an ODP2/BBM protein of the disclosurewill encode at least 15, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 709 contiguous amino acids, or up to thetotal number of amino acids present in a full-length ODP2/BBM protein ofthe disclosure. Fragments of an ODP2 nucleotide sequence that are usefulas hybridization probes or PCR primers generally need not encode abiologically active portion of an ODP2/BBM protein.

Thus, a fragment of an ODP2/BBM nucleotide sequence may encode abiologically active portion of an ODP2/BBM protein, or it may be afragment that can be used as a hybridization probe or PCR primer usingmethods disclosed below. A biologically active portion of an ODP2/BBMprotein can be prepared by isolating a portion of one of the ODP2/BBMnucleotide sequences of the disclosure, expressing the encoded portionof the ODP2/BBM protein (e.g., by recombinant expression in vitro), andassessing the activity of the encoded portion of the ODP2/BBM protein.Nucleic acid molecules that are fragments of an ODP2/BBM nucleotidesequence comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200, 1,300,or 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200contiguous nucleotides, or up to the number of nucleotides present in afull-length ODP2/BBM nucleotide sequence disclosed herein (for example,SEQ ID NOS:17, 19, and 21).

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a deletion and/or addition of oneor more nucleotides at one or more internal sites within the nativepolynucleotide and/or a substitution of one or more nucleotides at oneor more sites in the native polynucleotide. As used herein, a “native”polynucleotide or polypeptide comprises a naturally occurring nucleotidesequence or amino acid sequence, respectively. For polynucleotides,conservative variants include those sequences that, because of thedegeneracy of the genetic code, encode the amino acid sequence of one ofthe ODP2/BBM polypeptides of the disclosure. Variant ODP2/BBM2polynucleotides also include synthetically derived polynucleotides, suchas those generated, for example, by using site-directed mutagenesis butwhich still encode an ODP2 protein of the disclosure. Generally,variants of a particular polynucleotide of the disclosure will have atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to thatparticular polynucleotide as determined by sequence alignment programsand parameters described elsewhere herein.

“Variant” protein is intended to mean a protein derived from the nativeprotein by deletion or addition of one or more amino acids at one ormore internal sites in the native protein and/or substitution of one ormore amino acids at one or more sites in the native protein. Variantproteins encompassed by the present disclosure are biologically active,that is they continue to possess the desired biological activity of thenative protein, that is, the polypeptide has ODP2/BBM activity (i.e.,modulating the regenerative capability of a plant, rendering the plantembryogenic, increasing the transformation efficiency of a plant,altering oil content of a plant, increasing cell proliferation,increasing abiotic stress tolerance, increasing or maintaining yieldunder abiotic stress, modifying starch content, increasing asexualembryo formation, binding DNA or regulating transcription) as describedherein. Such variants may result from, for example, genetic polymorphismor from human manipulation. Biologically active variants of a nativeODP2/BBM protein of the disclosure will have at least about 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to the amino acid sequencefor the native protein as determined by sequence alignment programs andparameters described elsewhere herein. A biologically active variant ofa protein of the disclosure may differ from that protein by as few as1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, asfew as 4, 3, 2, or even 1 amino acid residue.

The ODP2/BBM proteins of the disclosure may be altered in various waysincluding amino acid substitutions, deletions, truncations, fusions, andinsertions. Methods for such manipulations are generally known in theart. For example, amino acid sequence variants of the ODP2 proteins canbe prepared by mutations in the DNA. Methods for mutagenesis andnucleotide sequence alterations are well known in the art. See, forexample, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel etal. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192;Walker and Gaastra, eds. (1983) Techniques in Molecular Biology(MacMillan Publishing Company, New York) and the references citedtherein. Guidance as to appropriate amino acid substitutions that do notaffect biological activity of the protein of interest may be found inthe model of Dayhoff et al. (1978) Atlas of Protein Sequence andStructure (Natl. Biomed. Res. Found., Washington, D.C.), hereinincorporated by reference. Conservative substitutions, such asexchanging one amino acid with another having similar properties, may beoptimal.

III. WUS/WOX Homeobox Polypeptides

The methods of the present disclosure comprise polynucleotide sequencesand amino acid sequences of WUS/WOX homeobox polypeptides. The Wuschelprotein, designated hereafter as WUS, plays a key role in the initiationand maintenance of the apical meristem, which contains a pool ofpluripotent stem cells (Endrizzi, et al., (1996) Plant Journal10:967-979; Laux, et al., (1996) Development 122:87-96; and Mayer, etal., (1998) Cell 95:805-815). Arabidopsis plants mutant for the WUS genecontain stem cells that are misspecified and that appear to undergodifferentiation. WUS encodes a novel homeodomain protein whichpresumably functions as a transcriptional regulator (Mayer, et al.,(1998) Cell 95:805-815). The stem cell population of Arabidopsis shootmeristems is believed to be maintained by a regulatory loop between theCLAVATA (CLV) genes which promote organ initiation and the WUS genewhich is required for stem cell identity, with the CLV genes repressingWUS at the transcript level, and WUS expression being sufficient toinduce meristem cell identity and the expression of the stem cell markerCLV3 (Brand, et al., (2000) Science 289:617-619; Schoof, et al., (2000)Cell 100:635-644). Constitutive expression of WUS in Arabidopsis hasbeen shown to lead to adventitious shoot proliferation from leaves (inplanta) (Laux, T., Talk Presented at the XVI International BotanicalCongress Meeting, Aug. 1-7, 1999, St. Louis, Mo.).

In an aspect, the WUS/WOX homeobox polypeptide is a WUS1, WUS2, WUS3,WOX2A, WOX4. WOX5, or WOX9 polypeptide (van der Graaff et al., 2009,Genome Biology 10:248). The WUS/WOX homeobox polypeptide can be amonocot WUS/WOX homeobox polypeptide. In various aspects, WUS/WOXhomeobox polypeptide can be a barley, maize, millet, oats, rice, rye,Setaria sp., sorghum, sugarcane, switchgrass, triticale, turfgrass, orwheat WUS/WOX homeobox polypeptide. Alternatively, the WUS/WOX homeoboxpolypeptide can be a dicot WUS/WOX homeobox polypeptide. In particular,the present disclosure provides for isolated nucleic acid moleculescomprising nucleotide sequences encoding the amino acid sequences shownin SEQ ID NOS: 4, 6, 8, 10, 12, 14, and 16. Further provided arepolypeptides having an amino acid sequence encoded by a nucleic acidmolecule (SEQ ID NO: 3, 5, 7, 9, 11, 13, and 15) described herein, andfragments and variants thereof.

The WUS/WOX homeobox polypeptide can be derived from a plant that is amember of the family Poaceae. Non-limiting examples of suitable plantsfrom which the WUS/WOX homeobox polypeptide can be derived include graincrops, including, but not limited to, barley, maize (corn), oats, rice,rye, sorghum, wheat, millet, triticale; leaf and stem crops, including,but not limited to, bamboo, marram grass, meadow-grass, reeds, ryegrass,sugarcane; lawn grasses, ornamental grasses, and other grasses such asswitchgrass and turfgrass.

In a further aspect, the WUS/WOX homeobox polypeptide used in thedisclosed methods can be derived from any plant, including higherplants, e.g., classes of Angiospermae and Gymnospermae. Plants of thesubclasses of the Dicotylodenae and the Monocotyledonae are suitable.Suitable species may come from the family Acanthaceae, Alliaceae,Alstroemeriaceae, Amaryllidaceae, Apocynaceae, Arecaceae, Asteraceae,Berberidaceae, Bixaceae, Brassicaceae, Bromeliaceae, Cannabaceae,Caryophyllaceae, Cephalotaxaceae, Chenopodiaceae, Colchicaceae,Cucurbitaceae, Dioscoreaceae, Ephedraceae, Erythroxylaceae,Euphorbiaceae, Fabaceae, Lamiaceae, Linaceae, Lycopodiaceae, Malvaceae,Melanthiaceae. Musaceae, Myrtaceae, Nyssaceae, Papaveraceae, Pinaceae,Plantaginaceae, Poaceae, Rosaceae, Rubiaceae, Salicaceae, Sapindaceae,Solanaceae, Taxaceae, Theaceae, and Vitaceae.

Suitable species from which the WUS/WOX homeobox polypeptide can bederived include members of the genus Abelmoschus, Abies, Acer, Agrostis,Allium, Alstroemeria, Ananas, Andrographis, Andropogon, Artemisia,Arundo, Atropa. Berberis, Beta. Bixa, Brassica. Calendula, Camellia,Camptotheca, Cannabis, Capsicum, Carthamus, Catharanthus, Cephalotaxus,Chrysanthemum, Cinchona, Citrullus, Coffea, Colchicum, Coleus, Cucumis,Cucurbita, Cynodon, Datura, Dianthus, Digitalis, Dioscorea, Elaeis,Ephedra, Erianthus, Erythroxylum, Eucalyptus, Festuca, Fragaria,Galanthus, Glycine, Gossypium, Helianthus, Hevea, Hordeum, Hyoscyamus,Jatropha, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Lycopodium,Manihot, Medicago, Mentha, Miscanthus, Musa, Nicotiana, Oryza, Panicum,Papaver. Parthenium, Pennisetum, Petunia, Phalaris, Phleum, Pinus, Poa,Poinsettia, Populus, Rauwolfia, Ricinus, Rosa, Saccharum, Salix,Sanguinaria, Scopolia, Secale, Solanum, Sorghum, Spartina, Spinacea,Tanacetum. Taxus, Theobroma, Triticosecale, Triticum, Uniola, Veratrum,Vinca, Vitis, and Zea.

In a further aspect, the WUS/WOX homeobox polypeptide can be derivedfrom a plant that is important or interesting for agriculture,horticulture, biomass for the production of liquid fuel molecules andother chemicals, and/or forestry. Non-limiting examples include, forinstance, Panicum virgatum (switchgrass), Sorghum bicolor (sorghum,sudangrass), Miscanthus giganteus (miscanthus), Saccharum sp. (energycane), Populus balsamifera (poplar), Zea mays (corn), Glycine max(soybean), Brassica napus (canola), Triticum aestivum (wheat), Gossypiumhirsutum (cotton), Oryza sativa (rice), Helianthus annuus (sunflower),Medicago sativa (alfalfa), Beta vulgaris (sugarbeet), Pennisetum glaucum(pearl millet), Panicum spp., Sorghum spp., Miscanthus spp., Saccharumspp., Erianthus spp., Populus spp., Andropogon gerardii (big bluestem),Pennisetum purpureum (elephant grass), Phalaris arundinacea (reed canarygrass), Cynodon dactylon (bermudagrass), Festuca arundinacea (tallfescue), Spartina pectinata (prairie cord-grass), Arundo donax (giantreed), Secale cereale (rye), Salix spp. (willow), Eucalyptus spp.(eucalyptus), Triticosecale spp. (triticum-wheat X rye), Bamboo,Carthamus tinctorius (safflower), Jatropha curcas (jatropha), Ricinuscommunis (castor), Elaeis guineensis (palm), Linum usitatissimum (flax),Brassica juncea, Manihot esculenta (cassava), Lycopersicon esculentum(tomato), Lactuca sativa (lettuce), Musa paradisiaca (banana), Solanumtuberosum (potato), Brassica oleracea (broccoli, cauliflower,brusselsprouts), Camellia sinensis (tea), Fragaria ananassa(strawberry), Theobroma cacao (cocoa), Coffea arabica (coffee), Vitisvinifera (grape), Ananas comosus (pineapple), Capsicum annum (hot &sweet pepper), Allium cepa (onion), Cucumis melo (melon), Cucumissativus (cucumber), Cucurbita maxima (squash), Cucurbita moschata(squash), Spinacea oleracea (spinach), Citrullus lanatus (watermelon),Abelmoschus esculentus (okra), Solanum melongena (eggplant), Papaversomniferum (opium poppy), Papaver orientale, Taxus baccata, Taxusbrevifolia, Artemisia annua, Cannabis sativa, Camptotheca acuminate,Catharanthus roseus, Vinca rosea. Cinchona officinalis, Colchicumautumnale, Veratrum californica, Digitalis lanata, Digitalis purpurea,Dioscorea spp., Andrographis paniculata, Atropa belladonna, Daturastomonium, Berberis spp., Cephalotaxus spp., Ephedra sinica, Ephedraspp., Erythroxylum coca. Galanthus wornorii, Scopolia spp., Lycopodiumserratum (=Huperzia serrata), Lycopodium spp., Rauwolfia serpentina,Rauwolfia spp., Sanguinaria canadensis, Hyoscyamus spp., Calendulaofficinalis, Chrysanthemum parthenium, Coleus forskohlii, Tanacetumparthenium, Parthenium argentatum (guayule), Hevea spp. (rubber), Menthaspicata (mint), Mentha piperita (mint), Bixa orellana. Alstroemeriaspp., Rosa spp. (rose), Dianthus caryophyllus (carnation), Petunia spp.(petunia), Poinsettia pulcherrima (poinsettia), Nicotiana tabacum(tobacco), Lupinus albus (lupin), Uniola paniculata (oats), bentgrass(Agrostis spp.), Populus tremuloides (aspen), Pinus spp. (pine), Abiesspp. (fir), Acer spp. (maple), Hordeum vulgare (barley), Poa pratensis(bluegrass), Lolium spp. (ryegrass), Phleum pratense (timothy), andconifers. Of interest are plants grown for energy production, so calledenergy crops, such as cellulose-based energy crops like Panicum virgatum(switchgrass). Sorghum bicolor (sorghum, sudangrass), Miscanthusgiganteus (miscanthus), Saccharum sp. (energy cane), Populus balsamifera(poplar), Andropogon gerardii (big bluestem), Pennisetum purpureum(elephant grass), Phalaris arundinacea (reed canary grass), Cynodondactylon (bermudagrass), Festuca arundinacea (tall fescue), Spartinapectinata (prairie cord-grass), Medicago saliva (alfalfa), Arundo donax(giant reed), Secale cereale (rye), Salix spp. (willow), Eucalyptus spp.(eucalyptus), Triticosecale spp. (triticum-wheat X rye), and Bamboo; andstarch-based energy crops like Zea mays (corn) and Manihot esculenta(cassava); and sucrose-based energy crops like Saccharum sp. (sugarcane)and Beta vulgaris (sugarbeet); and biodiesel-producing energy crops likeGlycine max (soybean), Brassica napus (canola), Helianthus annuus(sunflower), Carthamus tinctorius (safflower), Jatropha curcas(jatropha), Ricinus communis (castor), Elaeis guineensis (palm), Linumusitatissimum (flax), and Brassica juncea.

The WUS/WOX homeobox polypeptide can be derived from a biomass renewableenergy source. Examples of biomass renewable energy source plantsinclude those described herein above.

The WUS/WOX homeobox polypeptide used in the disclosed methods andcompositions can be a WUS1 polypeptide. In an aspect, the WUS1polypeptide is a maize, sorghum, rice or Setaria sp. WUS1 polypeptide.In a further aspect, the WUS1 polypeptide is a maize or rice WUS1polypeptide. In a further aspect, the WUS1 polypeptide is a maize WUS1polypeptide. In particular, the WUS1 polypeptide comprises an amino acidsequence of SEQ ID NO:4 or the WUS1 polypeptide is encoded by anucleotide sequence comprising SEQ ID NO:3.

The WUS/WOX homeobox polypeptide used in the disclosed methods andcompositions can be a WUS2 polypeptide. In various aspects, the WUS2polypeptide is a barley, maize, millet, oats, rice, rye, Setaria sp.,sorghum, sugarcane, switchgrass, triticale, turfgrass, or wheat WUSpolypeptide. In an aspect, the WUS2 polypeptide is a maize, sorghum,rice or Setaria sp. WUS2 polypeptide. In a further aspect, the WUS2polypeptide is a maize or rice WUS2 polypeptide. In a further aspect,the WUS2 polypeptide is a maize WUS2 polypeptide. In particular, theWUS2 polypeptide comprises an amino acid sequence of SEQ ID NO:6 or theWUS2 polypeptide is encoded by a nucleotide sequence comprising SEQ IDNO:5.

The WUS/WOX homeobox polypeptide used in the disclosed methods andcompositions can be a WUS3 polypeptide. In an aspect, The WUS3polypeptide is a maize, sorghum, rice or Setaria sp. WUS3 polypeptide.In a further aspect, the WUS3 polypeptide is a maize or rice WUS3polypeptide. In a further aspect, the WUS3 polypeptide is a maize WUS3polypeptide. In particular, the WUS3 polypeptide comprises an amino acidsequence of SEQ ID NO:8 or the WUS3 polypeptide is encoded by anucleotide sequence comprising SEQ ID NO:7.

The WUS/WOX homeobox polypeptide used in the disclosed methods andcompositions can be a WOX2A polypeptide. In an aspect, the WOX2Apolypeptide is a maize, sorghum, rice or Setaria sp. WOX2A polypeptide.In a further aspect, the WOX2A polypeptide is a maize or rice WUS2Apolypeptide. In a further aspect, the WOX2A polypeptide is a maize WOX2Apolypeptide. In particular, the WOX2A polypeptide comprises an aminoacid sequence of SEQ ID NO: 10 or the WOX2A polypeptide is encoded by anucleotide sequence comprising SEQ ID NO:9.

The WUS/WOX homeobox polypeptide used in the disclosed methods andcompositions can be a WOX4 polypeptide. In an aspect, the WOX4polypeptide is a maize, sorghum, rice or Setaria sp. WOX4 polypeptide.In a further aspect, the WOX4 polypeptide is a maize or rice WUS4polypeptide. In a further aspect, the WOX4 polypeptide is a maize WOX4polypeptide. In particular, the WOX4 polypeptide comprises an amino acidsequence of SEQ ID NO:12 or the WOX4 polypeptide is encoded by anucleotide sequence comprising SEQ ID NO:11.

The WUS/WOX homeobox polypeptide used in the disclosed methods andcompositions can be a WOX5A polypeptide. In an aspect, the WOX5Apolypeptide is a maize, sorghum, rice or Setaria sp. WOX5A polypeptide.In a further aspect, the WOX5A polypeptide is a maize or rice WUS5Apolypeptide. In a further aspect, the WOX5A polypeptide is a maize WOX5Apolypeptide. In particular, the WOX5A polypeptide comprises an aminoacid sequence of SEQ ID NO:14 or the WOX5A polypeptide is encoded by anucleotide sequence comprising SEQ ID NO: 13.

The WUS/WOX homeobox polypeptide used in the disclosed methods andcompositions can be a WOX9 polypeptide. In an aspect, The WOX9polypeptide is a maize, sorghum, rice or Setaria sp. WOX9 polypeptide.In a further aspect, the WOX9 polypeptide is a maize or rice WUS9polypeptide. In a further aspect, the WOX9 polypeptide is a maize WOX9polypeptide. In particular, the WOX9 polypeptide comprises an amino acidsequence of SEQ ID NO:16 or the WOX9 polypeptide is encoded by anucleotide sequence comprising SEQ ID NO:15.

The present disclosure encompasses isolated or substantially purifiednucleic acid or protein WUS/WOX compositions. Fragments and variants ofthe WUS/WOX nucleotide sequences and proteins encoded thereby are alsoencompassed by the present disclosure. By “fragment” is intended aportion of the nucleotide sequence or a portion of the amino acidsequence and hence protein encoded thereby. Fragments of a nucleotidesequence may encode protein fragments that retain the biologicalactivity of the native protein and hence have WUS/WOX activity.Alternatively, fragments of a nucleotide sequence useful ashybridization probes generally do not encode fragment proteins retainingbiological activity. Thus, fragments of a nucleotide sequence may rangefrom at least about 20 nucleotides, about 50 nucleotides, about 100nucleotides, and up to the full-length nucleotide sequence encoding theproteins of the disclosure.

A fragment of a WUS/WOX nucleotide sequence that encodes a biologicallyactive portion of a WUS/WOX protein of the disclosure will encode atleast 15, 25, 30, 50, 10, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 709 contiguous amino acids, or up to the total number ofamino acids present in a full-length WUS/WOX protein of the disclosure.Fragments of a WUS/WOX nucleotide sequence useful as hybridizationprobes or PCR primers generally need not encode a biologically activeportion of a WUS/WOX protein.

Thus, a fragment of a WUS/WOX nucleotide sequence may encode abiologically active portion of a WUS/WOX protein, or it may be afragment that can be used as a hybridization probe or PCR primer usingmethods disclosed below. A biologically active portion of a WUS/WOXprotein can be prepared by isolating a portion of one of the WUS/WOXnucleotide sequences of the disclosure, expressing the encoded portionof the WUX/WOX protein (e.g., by recombinant expression in vitro), andassessing the activity of the encoded portion of the WUS/WOX protein.Nucleic acid molecules that are fragments of a WUS/WOX nucleotidesequence comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200, 1,300,or 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200contiguous nucleotides, or up to the number of nucleotides present in afull-length WUS/WOX nucleotide sequence disclosed herein (for example,SEQ ID NOS: 3, 5, 7, 9, 11, 13, and 15).

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, conservative variants include those sequences that,because of the degeneracy of the genetic code, encode the amino acidsequence of one of the WUS/WOX polypeptides of the disclosure. Variantpolynucleotides also include synthetically derived polynucleotides, suchas those generated, for example, by using site-directed mutagenesis butwhich still encode a WUS/WOX protein of the disclosure. Generally,variants of a particular polynucleotide of the disclosure will have atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to thatparticular polynucleotide as determined by sequence alignment programsand parameters described elsewhere herein.

“Variant” protein is intended to mean a protein derived from the nativeprotein by deletion or addition of one or more amino acids at one ormore internal sites in the native protein and/or substitution of one ormore amino acids at one or more sites in the native protein. Variantproteins encompassed by the present disclosure are biologically active,that is they continue to possess the desired biological activity of thenative protein, that is, the polypeptide has WUS/WOX activity. Suchvariants may result from, for example, genetic polymorphism or fromhuman manipulation. Biologically active variants of a native WUS/WOXprotein of the disclosure will have at least about 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity to the amino acid sequence for thenative protein as determined by sequence alignment programs andparameters described elsewhere herein. A biologically active variant ofa protein of the disclosure may differ from that protein by as few as1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, asfew as 4, 3, 2, or even 1 amino acid residue.

The WUS/WOX proteins of the disclosure may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions. Methods for such manipulations are generally known in theart. For example, amino acid sequence variants of the WUS/WOX proteinscan be prepared by mutations in the DNA. Methods for mutagenesis andnucleotide sequence alterations are well known in the art. See, forexample, Kunkel (1985)Proc. Natl. Acad Sci. USA 82:488-492; Kunkel etal. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192;Walker and Gaastra, eds. (1983) Techniques in Molecular Biology(MacMillan Publishing Company, New York) and the references citedtherein. Guidance as to appropriate amino acid substitutions that do notaffect biological activity of the protein of interest may be found inthe model of Dayhoff et al. (1978) Atlas of Protein Sequence andStructure (Natl. Biomed. Res. Found., Washington, D.C.), hereinincorporated by reference. Conservative substitutions, such asexchanging one amino acid with another having similar properties, may beoptimal.

IV. Transformation Methods

The methods and composition of the disclosure can utilize a variety oftransformation methods as appropriate. That is, transformation protocolsas well as protocols for introducing nucleotide sequences into plantsmay vary depending on the type of plant or plant cell, i.e., monocot ordicot, targeted for transformation. Suitable methods of introducingnucleotide sequences into plant cells and subsequent insertion into theplant genome include microinjection (Crossway et al. (1986)Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc.Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediated transformation(Townsend et al., U.S. Pat. No. 5,563,055; Zhao et al., U.S. Pat. No.5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J3:2717-2722), and ballistic particle acceleration (see, for example,Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al., U.S. Pat. No.5,879,918; Tomes et al., U.S. Pat. No. 5,886,244; Bidney et al., U.S.Pat. No. 5,932,782; Tomes et al. (1995) “Direct DNA Transfer into IntactPlant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, andOrgan Culture: Fundamental Methods, ed. Gamborg and Phillips(Springer-Verlag, Berlin); and McCabe et al. (1988) Biotechnology6:923-926). Also see Weissinger et al. (1988) Ann. Rev. Genet.22:421-477; Sanford et al. (1987) Particulate Science and Technology5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674(soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean);Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182(soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean);Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988)Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988)Biotechnology 6:559-563 (maize); Tomes, U.S. Pat. No. 5,240,855; Buisinget al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomes et al. (1995)“Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment,” in Plant Cell. Tissue, and Organ Culture: FundamentalMethods, ed. Gamborg (Springer-Verlag, Berlin) (maize); Klein et al.(1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990)Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984)Nature (London) 311:763-764; Bowen et al., U.S. Pat. No. 5,736,369(cereals); Bytebier et al. (1987)Proc. Natl. Acad. Sci. USA 84:5345-5349(Liliaceae); De Wet et al. (1985) in The Experimental Manipulation ofOvule Tissues, ed. Chapman et al. (Longman, New York), pp. 197-209(pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 andKaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediatedtransformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505(electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 andChristou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda etal. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacteriumtumefaciens); all of which are herein incorporated by reference.

The methods and composition of the present disclosure may be used fortransformation of any plant species, including, but not limited to,monocots and dicots. Examples of plant species of interest include, butare not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus. B.rapa, B. juncea), particularly those Brassica species useful as sourcesof seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secalecereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g.,pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum),foxtail millet (Setaria italica), finger millet (Eleusine coracana)),sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat(Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum),potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton(Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoeabatatus), cassaya (Manihot esculenta), coffee (Coffea spp.), coconut(Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrusspp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musaspp.), avocado (Persea americana), fig (Ficus casica), guava (Psidiumguajava), mango (Mangifera indica), olive (Olea europaea), papaya(Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamiaintegrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris),sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, andconifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum.

Conifers that may be employed in practicing the present disclosureinclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Eastern or Canadian hemlock (Tsuga canadensis);Western hemlock (Tsuga heterophylla); Mountain hemlock (Tsugamertensiana); Tamarack or Larch (Larix occidentalis); Sitka spruce(Picea glauca); redwood (Sequoia sempervirens); true firs such as silverfir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such asWestern red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparisnootkatensis). Eucalyptus species may be employed in practicing thepresent disclosure, including E. grandis (and its hybrids, as“urograndis”), E. globulus, E. camaldulensis, E. tereticornis, E.viminalis, E. nitens, E. saligna and E. urophylla. Optimally, plants ofthe present disclosure are crop plants (for example, corn, alfalfa,sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat,millet, tobacco, etc.), more optimally corn and soybean plants, yet moreoptimally corn plants.

Plants of particular interest include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants include,but are not limited to, beans and peas. Beans include, but are notlimited to, guar, locust bean, fenugreek, soybean, garden beans, cowpea,mungbean, lima bean, fava bean, lentils, and chickpea.

A selectable marker comprises a DNA segment that allows one to identifyor select for or against a molecule or a cell that contains it, oftenunder particular conditions. These markers can encode an activity, suchas, but not limited to, production of RNA, peptide, or protein, or canprovide a binding site for RNA, peptides, proteins, inorganic andorganic compounds or compositions and the like. Examples of selectablemarkers include, but are not limited to, DNA segments that compriserestriction enzyme sites; DNA segments that encode products whichprovide resistance against otherwise toxic compounds (e.g., antibiotics,such as, spectinomycin, ampicillin, kanamycin, tetracycline, Basta,neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase(HPT)); DNA segments that encode products which are otherwise lacking inthe recipient cell (e.g., tRNA genes, auxotrophic markers); DNA segmentsthat encode products which can be readily identified (e.g., phenotypicmarkers such as β-galactosidase. GUS; fluorescent proteins such as greenfluorescent protein (GFP), cyan (CFP), yellow (YFP), red (RFP), and cellsurface proteins); the generation of new primer sites for PCR (e.g., thejuxtaposition of two DNA sequence not previously juxtaposed), theinclusion of DNA sequences not acted upon or acted upon by a restrictionendonuclease or other DNA modifying enzyme, chemical, etc.; and, theinclusion of a DNA sequences required for a specific modification (e.g.,methylation) that allows its identification.

Additional selectable markers include genes that confer resistance toherbicidal compounds, such as glyphosate, sulfonylureas, glufosinateammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate(2,4-D). See generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511;Christopherson et al. (1992) Proc. Natl. Acad Sci. USA 89:6314-6318; Yaoet al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol.6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al.(1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge etal. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Acad Sci.USA 86:5400-5404, Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993)Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl.Acad Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol.10:3343-3356; Zambretti et al. (1992)Proc. Natl. Acad Sci. USA89:3952-3956; Baim et al. (1991) Proc. Natl. Acad Sci. USA 88:5072-5076;Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653; Hillen andWissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolb et al.(1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidt et al.(1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis, Universityof Heidelberg; Gossen et al. (1992) Proc. Natl. Acad Sci. USA89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother.36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology,Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature334:721-724. Such disclosures are herein incorporated by reference. Theabove list of selectable markers is not meant to be limiting. Anyselectable marker can be used in the methods and compositions.

Because the timeframe of somatic embryogenesis and embryo maturation forthe described method is so truncated, the selection regime accordinglymust occur rapidly in order to effectively eliminate non-transgenicembryos and the resultant germinated T0 plants. As a result, dependingon the stringency of the selective agent over short timeframes, thepercentage of recovered “escapes” (or regenerated T0 plants that arewild-type) increase. To eliminate escapes, traditional in vitroselection of transgenic events in cereals has occurred over a timeframeof 1.5 to 3 months with multiple subcultures on selection medium beingrequired to amass enough callus tissue for regeneration (Gordon-Kamm etal., 1990, Plant Cell 2:603-618; Negrotto et al., 2000, Plant CellReports; Zhao et al., 2001, Mol. Breed. 8, 323-333; Wu et al., 2014, InVitro Cell. Dev. Biol. 50:9-18; see Kan Wang, 2014, AgrobacteriumProtocols, Vol. 1, third edition, Springer-Verlag, New York for review).The present disclosure eliminates much of this long-duration selectionby eliminating callus culture and instead progressing directly throughrapid embryogenesis and germination into independent T0 plants (eachrepresenting a separate integration event), requiring selective agentsthat work rapidly.

Certain selectable markers useful in the present method include, but arenot limited to, the maize HRA gene (Lee et al., 1988, EMBO J7:1241-1248) which confers resistance to sulfonylureas andimidazolinones, the GAT gene which confers resistance to glyphosate(Castle et al., 2004, Science 304:1151-1154), genes that conferresistance to spectinomycin such as the aadA gene (Svab et al., 1990,Plant Mol Biol. 14:197-205) and the bar gene that confers resistance toglufosinate ammonium (White et al., 1990, Nucl. Acids Res. 25:1062), andPAT (or moPAT for corn, see Rasco-Gaunt et al., 2003, Plant Cell Rep.21:569-76) and the PMI gene that permits growth on mannose-containingmedium (Negrotto et al., 2000, Plant Cell Rep. 22:684-690) are veryuseful for rapid selection during the brief elapsed time encompassed bysomatic embryogenesis and embryo maturation of the method. However,depending on the selectable marker used and the crop, inbred or varietybeing transformed, the percentage of wild-type escapes can vary. Inmaize and sorghum the HRA gene is efficacious in reducing the frequencyof wild-type escapes.

V. Methods to Improve Plant Traits and Characteristics

The present disclosure provides novel compositions and methods forproducing transgenic plants with increased efficiency and speed. Thedisclosed methods and compositions can further comprise polynucleotidesthat provide for improved traits and characteristics.

As used herein, “trait” refers to a physiological, morphological,biochemical, or physical characteristic of a plant or particular plantmaterial or cell. In some instances, this characteristic is visible tothe human eye, such as seed or plant size, or can be measured bybiochemical techniques, such as detecting the protein, starch, or oilcontent of seed or leaves, or by observation of a metabolic orphysiological process, e.g. by measuring uptake of carbon dioxide, or bythe observation of the expression level of a gene or genes, e.g., byemploying Northern analysis. RT-PCR, microarray gene expression assays,or reporter gene expression systems, or by agricultural observationssuch as stress tolerance, yield, or pathogen tolerance. An “enhancedtrait” as used in describing the aspects of the present disclosureincludes improved or enhanced water use efficiency or drought tolerance,osmotic stress tolerance, high salinity stress tolerance, heat stresstolerance, enhanced cold tolerance, including cold germinationtolerance, increased yield, enhanced nitrogen use efficiency, earlyplant growth and development, late plant growth and development,enhanced seed protein, and enhanced seed oil production.

Any polynucleotide of interest can be used in the methods of thedisclosure. Various changes in phenotype are of interest includingmodifying the fatty acid composition in a plant, altering the amino acidcontent, starch content, or carbohydrate content of a plant, altering aplant's pathogen defense mechanism, affecting kernel size, sucroseloading, and the like. The gene of interest may also be involved inregulating the influx of nutrients, and in regulating expression ofphytate genes particularly to lower phytate levels in the seed. Theseresults can be achieved by providing expression of heterologous productsor increased expression of endogenous products in plants. Alternatively,the results can be achieved by providing for a reduction of expressionof one or more endogenous products, particularly enzymes or cofactors inthe plant. These changes result in a change in phenotype of thetransformed plant.

Genes of interest are reflective of the commercial markets and interestsof those involved in the development of the crop. Crops and markets ofinterest change, and as developing nations open up world markets, newcrops and technologies will emerge also. In addition, as ourunderstanding of agronomic traits and characteristics such as yield andheterosis increase, the choice of genes for transformation will changeaccordingly. General categories of genes of interest include, forexample, those genes involved in information, such as zinc fingers,those involved in communication, such as kinases, and those involved inhousekeeping, such as heat shock proteins. More specific categories oftransgenes, for example, include genes encoding important traits foragronomics, insect resistance, disease resistance, herbicide resistance,sterility, grain characteristics, and commercial products. Genes ofinterest include, generally, those involved in oil, starch,carbohydrate, or nutrient metabolism as well as those affecting kernelsize, sucrose loading, and the like.

The polynucleotides introduced into an explant by the disclosed methodsand compositions can be operably linked to a suitable promoter.“Promoter” means a region of DNA that is upstream from the start oftranscription and is involved in recognition and binding of RNApolymerase and other proteins to initiate transcription, eitherincluding or not including the 5′ UTR. A “plant promoter” is a promotercapable of initiating transcription in plant cells whether or not itsorigin is a plant cell. Exemplary plant promoters include, but are notlimited to, those that are obtained from plants, plant viruses, andbacteria which comprise genes expressed in plant cells such as fromAgrobacterium or Rhizobium. Examples of promoters under developmentalcontrol include promoters that preferentially initiate transcription incertain tissues, such as leaves, roots, or seeds. Such promoters arereferred to as “tissue preferred”. Promoters which initiatetranscription only in certain tissues are referred to as “tissuespecific”. A “cell type” specific promoter primarily drives expressionin certain cell types in one or more organs, for example, vascular cellsin roots or leaves. An “inducible” or “repressible” promoter can be apromoter which is under either environmental or exogenous control.Examples of environmental conditions that may effect transcription byinducible promoters include anaerobic conditions, or certain chemicals,or the presence of light. Alternatively, exogenous control of aninducible or repressible promoter can be affected by providing asuitable chemical or other agent that via interaction with targetpolypeptides result in induction or repression of the promoter. Tissuespecific, tissue preferred, cell type specific, and inducible promotersconstitute the class of “non-constitutive” promoters. A “constitutive”promoter is a promoter which is active under most conditions. As usedherein, “antisense orientation” includes reference to a polynucleotidesequence that is operably linked to a promoter in an orientation wherethe antisense strand is transcribed. The antisense strand issufficiently complementary to an endogenous transcription product suchthat translation of the endogenous transcription product is ofteninhibited. “Operably linked” refers to the association of two or morenucleic acid fragments on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

Agronomically important traits such as oil, starch, and protein contentcan be genetically altered in addition to using traditional breedingmethods. Modifications include increasing content of oleic acid,saturated and unsaturated oils, increasing levels of lysine and sulfur,providing essential amino acids, and also modification of starch.Hordothionin protein modifications are described in U.S. Pat. Nos.5,703,049, 5,885,801, 5,885,802, and 5,990,389, herein incorporated byreference. Another example is lysine and/or sulfur rich seed proteinencoded by the soybean 2S albumin described in U.S. Pat. No. 5,850,016,and the chymotrypsin inhibitor from barley, described in Williamson etal. (1987) Eur. J Biochem. 165:99-106, the disclosures of which areherein incorporated by reference.

Derivatives of the coding sequences can be made by site-directedmutagenesis to increase the level of preselected amino acids in theencoded polypeptide. For example, methionine-rich plant proteins such asfrom sunflower seed (Lilley et al. (1989) Proceedings of the WorldCongress on Vegetable Protein Utilization in Human Foods and AnimalFeedstuffs, ed. Applewhite (American Oil Chemists Society, Champaign,Ill.). pp. 497-502; herein incorporated by reference); corn (Pedersen etal. (1986) J Biol. Chem. 261:6279; Kirihara et al. (1988) Gene 71:359;both of which are herein incorporated by reference); and rice (Musumuraet al. (1989) Plant Mol. Biol. 12:123, herein incorporated by reference)could be used. Other agronomically important genes encode latex, Floury2, growth factors, seed storage factors, and transcription factors.

Insect resistance genes may encode resistance to pests that have greatyield drag such as rootworm, cutworm, European Corn Borer, and the like.Such genes include, for example, Bacillus thuringiensis toxic proteingenes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756;5,593,881; and Geiser et al. (1986) Gene 48:109); and, the like.

Genes encoding disease resistance traits include detoxification genes,such as against fumonosin (U.S. Pat. No. 5,792,931); avirulence (avr)and disease resistance (R) genes (Jones et al. (1994) Science 266:789;Martin et al. (1993) Science 262:1432; and Mindrinos et al. (1994) Cell78:1089); and the like.

Herbicide resistance traits may include genes coding for resistance toherbicides that act to inhibit the action of acetolactate synthase(ALS), in particular the sulfonylurea-type herbicides (e.g., theacetolactate synthase (ALS) gene containing mutations leading to suchresistance, in particular the S4 and/or Hra mutations), genes coding forresistance to herbicides that act to inhibit action of glutaminesynthase, such as phosphinothricin or basta (e.g., the bar gene),glyphosate (e.g., the EPSPS gene and the GAT gene; see, for example,U.S. Publication No. 20040082770 and WO 03/092360) or other such genesknown in the art. The bar gene encodes resistance to the herbicidebasta, the nptII gene encodes resistance to the antibiotics kanamycinand geneticin, and the ALS-gene mutants encode resistance to theherbicide chlorsulfuron.

Sterility genes can also be encoded in an expression cassette andprovide an alternative to physical detasseling. Examples of genes usedin such ways include male tissue-preferred genes and genes with malesterility phenotypes such as QM, described in U.S. Pat. No. 5,583,210.Other genes include kinases and those encoding compounds toxic to eithermale or female gametophytic development.

The quality of grain is reflected in traits such as levels and types ofoils, saturated and unsaturated, quality and quantity of essential aminoacids, and levels of cellulose. In corn, modified hordothionin proteinsare described in U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802, and5,990,389.

Commercial traits can also be encoded on a gene or genes that couldincrease for example, starch for ethanol production, or provideexpression of proteins. Another important commercial use of transformedplants is the production of polymers and bioplastics such as describedin U.S. Pat. No. 5,602,321. Genes such as D-Ketothiolase, PHBase(polyhydroxyburyrate synthase), and acetoacetyl-CoA reductase (seeSchubert et al. (1988) J. Bacteriol. 170:5837-5847) facilitateexpression of polyhyroxyalkanoates (PHAs).

Exogenous products include plant enzymes and products as well as thosefrom other sources including prokaryotes and other eukaryotes. Suchproducts include enzymes, cofactors, hormones, and the like. The levelof proteins, particularly modified proteins having improved amino aciddistribution to improve the nutrient value of the plant, can beincreased. This is achieved by the expression of such proteins havingenhanced amino acid content.

In an aspect, further agronomic traits of interest that can beintroduced into explants with increased efficiency and speed are suchtraits as increased yield or other trait that provides increased plantvalue, including, for example, improved seed quality. Of particularinterest are traits that provide improved or enhanced water useefficiency or drought tolerance, osmotic stress tolerance, high salinitystress tolerance, heat stress tolerance, enhanced cold tolerance,including cold germination tolerance, increased yield, enhanced nitrogenuse efficiency, early plant growth and development, late plant growthand development, enhanced seed protein, and enhanced seed oilproduction.

Many agronomic traits can affect “yield”, including without limitation,plant height, pod number, pod position on the plant, number ofinternodes, incidence of pod shatter, grain size, efficiency ofnodulation and nitrogen fixation, efficiency of nutrient assimilation,resistance to biotic and abiotic stress, carbon assimilation, plantarchitecture, resistance to lodging, percent seed germination, seedlingvigor, and juvenile traits. Other traits that can affect yield include,efficiency of germination (including germination in stressedconditions), growth rate (including growth rate in stressed conditions),ear number, seed number per ear, seed size, composition of seed (starch,oil, protein) and characteristics of seed fill. Also of interest is thegeneration of transgenic plants that demonstrate desirable phenotypicproperties that may or may not confer an increase in overall plantyield. Such properties include enhanced plant morphology, plantphysiology or improved components of the mature seed harvested from thetransgenic plant.

“Increased yield” of a transgenic plant of the present disclosure may beevidenced and measured in a number of ways, including test weight, seednumber per plant, seed weight, seed number per unit area (i.e. seeds, orweight of seeds, per acre), bushels per acre, tons per acre, kilo perhectare. For example, maize yield may be measured as production ofshelled corn kernels per unit of production area, e.g. in bushels peracre or metric tons per hectare, often reported on a moisture adjustedbasis, e.g., at 15.5% moisture. Increased yield may result from improvedutilization of key biochemical compounds, such as nitrogen, phosphorousand carbohydrate, or from improved tolerance to environmental stresses,such as cold, heat, drought, salt, and attack by pests or pathogens.Trait-enhancing recombinant DNA may also be used to provide transgenicplants having improved growth and development, and ultimately increasedyield, as the result of modified expression of plant growth regulatorsor modification of cell cycle or photosynthesis pathways.

Many agronomic traits can affect “yield”, including without limitation,plant height, pod number, pod position on the plant, number ofinternodes, incidence of pod shatter, grain size, efficiency ofnodulation and nitrogen fixation, efficiency of nutrient assimilation,resistance to biotic and abiotic stress, carbon assimilation, plantarchitecture, resistance to lodging, percent seed germination, seedlingvigor, and juvenile traits. Other traits that can affect yield include,efficiency of germination (including germination in stressedconditions), growth rate (including growth rate in stressed conditions),ear number, seed number per ear, seed size, composition of seed (starch,oil, protein) and characteristics of seed fill. Also of interest is thegeneration of transgenic plants that demonstrate desirable phenotypicproperties that may or may not confer an increase in overall plantyield. Such properties include enhanced plant morphology, plantphysiology or improved components of the mature seed harvested from thetransgenic plant.

VI. Methods to Suppress Genes

In an aspect, the disclosed methods and compositions can be used tointroduce into plants with increased efficiency and speedpolynucleotides useful for gene suppression of a target gene in a plant.Reduction of the activity of specific genes (also known as genesilencing, or gene suppression) is desirable for several aspects ofgenetic engineering in plants. Many techniques for gene silencing arewell known to one of skill in the art, including but not limited toantisense technology (see, e.g., Sheehy et al. (1988) Proc. Natl. Acad.Sci. USA 85:8805-8809; and U.S. Pat. Nos. 5,107,065; 5,453,566; and5,759,829); cosuppression (e.g., Taylor (1997) Plant Cell 9:1245;Jorgensen (1990) Trends Biotech. 8(12):340-344; Flavell (1994) Proc.Natl. Acad. Sci. USA 91:3490-3496; Finnegan et al. (1994) Bio-Technology12: 883-888; and Neuhuber et al. (1994) Mol. Gen. Genet. 244:230-241);RNA interference (Napoli et al. (1990) Plant Cell 2:279-289; U.S. Pat.No. 5,034,323; Sharp (1999) Genes Dev. 13:139-141; Zamore et al. (2000)Cell 101:25-33; Javier (2003) Nature 425:257-263; and, Montgomery et al.(1998) Proc. Natl. Acad. Sci. USA 95:15502-15507), virus-induced genesilencing (Burton, et al. (2000) Plant Cell 12:691-705; and Baulcombe(1999) Curr. Op. Plant Bio. 2:109-113); target-RNA-specific ribozymes(Haseloff et al. (1988) Nature 334: 585-591); hairpin structures (Smithet al. (2000) Nature 407:319-320; WO 99/53050; WO 02/00904; and WO98/53083); ribozymes (Steinecke et al. (1992) EMBO J. 11:1525; U.S. Pat.No. 4,987,071, and, Perriman et al. (1993) Antisense Res. Dev. 3:253);oligonucleotide mediated targeted modification (e.g., WO 03/076574 andWO 99/25853); Zn-finger targeted molecules (e.g., WO 01/52620; WO03/048345; and WO 00/42219); artificial micro RNAs (U.S. Pat. No.8,106,180; Schwab et al. (2006) Plant Cell 18:1121-1133); and othermethods or combinations of the above methods known to those of skill inthe art.

VII. Methods to Introduce Genome Editing Technologies into Plants

In an aspect, the disclosed methods and compositions can be used tointroduce into plants with increased efficiency and speedpolynucleotides useful to target a specific site for modification in thegenome of a plant. Site specific modifications that can be introducedwith the disclosed methods and compositions include those produced usingany method for introducing site specific modification, including, butnot limited to, through the use of gene repair oligonucleotides (e.g. USPublication 2013/0019349), or through the use of double-stranded breaktechnologies such as TALENs, meganucleases, zinc finger nucleases,CRISPR-Cas, and the like. For example, the disclosed methods andcompositions can be used to introduce a CRISPR-Cas system into plants,for the purpose of genome modification of a target sequence in thegenome of a plant or plant cell, for selecting plants, for deleting abase or a sequence, for gene editing, and for inserting a polynucleotideof interest into the genome of a plant. Thus, the disclosed methods andcompositions can be used together with a CRISPR-Cas system to providefor an effective system for modifying or altering target sites andnucleotides of interest within the genome of a plant, plant cell orseed.

In an aspect, the present disclosure comprises methods and compositionsfor producing a transgenic plant, wherein the method comprisesintroducing a polynucleotide of interest into a target site in thegenome of a plant cell, the method comprising (a) transforming one ormore cells of an explant with an expression construct comprising: (i) anucleotide sequence encoding a WUS/WOX homeobox polypeptide; (ii) anucleotide sequence encoding a polypeptide comprising two AP2-DNAbinding domains; or (iii) a combination of (i) and (ii); and (b)allowing expression of the polypeptide of (a) in each transformed cellto form a regenerable plant structure, in the absence of cytokinin,wherein no callus is formed; and wherein transformation furthercomprises a first expression construct capable of expressing a guidenucleotide and a second recombinant DNA construct capable of expressinga Cas endonuclease, wherein the guide nucleotide and Cas endonucleaseare capable of forming a complex that enables the Cas endonuclease tointroduce a double strand break at the target site. Alternatively, theexpression construct comprising the nucleotide sequence encoding aWUS/WOX homeobox polypeptide and/or nucleotide sequence encoding apolypeptide comprising two AP2-DNA binding domains can also comprise anucleotide sequence capable of expressing the guide nucleotide and anucleotide sequence capable of expressing the Cas endonuclease.

In an aspect, the Cas endonuclease gene is a plant optimized Cas9endonuclease, wherein the plant optimized Cas9 endonuclease is capableof binding to and creating a double strand break in a genomic targetsequence the plant genome.

The Cas endonuclease is guided by the guide nucleotide to recognize andoptionally introduce a double strand break at a specific target siteinto the genome of a cell. The CRISPR-Cas system provides for aneffective system for modifying target sites within the genome of aplant, plant cell or seed. Further provided are methods and compositionsemploying a guide polynucleotide/Cas endonuclease system to provide aneffective system for modifying target sites within the genome of a celland for editing a nucleotide sequence in the genome of a cell. Once agenomic target site is identified, a variety of methods can be employedto further modify the target sites such that they contain a variety ofpolynucleotides of interest. The disclosed compositions and methods canbe used to introduce a CRISPR-Cas system for editing a nucleotidesequence in the genome of a cell. The nucleotide sequence to be edited(the nucleotide sequence of interest) can be located within or outside atarget site that is recognized by a Cas endonuclease.

CRISPR loci (Clustered Regularly Interspaced Short Palindromic Repeats)(also known as SPIDRs-SPacer Interspersed Direct Repeats) constitute afamily of recently described DNA loci. CRISPR loci consist of short andhighly conserved DNA repeats (typically 24 to 40 bp, repeated from 1 to140 times—also referred to as CRISPR-repeats) which are partiallypalindromic. The repeated sequences (usually specific to a species) areinterspaced by variable sequences of constant length (typically 20 to 58by depending on the CRISPR locus (WO2007/025097 published Mar. 1, 2007).

CRISPR loci were first recognized in E. coli (Ishino et al. (1987) J.Bacterial. 169:5429-5433; Nakata et al. (1989) J. Bacterial.171:3553-3556). Similar interspersed short sequence repeats have beenidentified in Haloferax mediterranei, Streptococcus pyogenes, Anabaena,and Mycobacterium tuberculosis (Groenen et al. (1993) Mol. Microbiol.10:1057-1065; Hoe et al. (1999) Emerg. Infect. Dis. 5:254-263; Masepohlet al. (19%) Biochim. Biophys. Acta 1307:26-30; Mojica et al. (1995)Mol. Microbiol. 17:85-93). The CRISPR loci differ from other SSRs by thestructure of the repeats, which have been termed short regularly spacedrepeats (SRSRs) (Janssen et al. (2002) OMICS J. Integ. Biol. 6:23-33;Mojica et al. (200(0) Mol. Microbiol. 36:244-246). The repeats are shortelements that occur in clusters, that are always regularly spaced byvariable sequences of constant length (Mojica et al. (2000) Mol.Microbiol. 36:244-246).

Cas gene includes a gene that is generally coupled, associated or closeto or in the vicinity of flanking CRISPR loci. The terms “Cas gene” and“CRISPR-associated (Cas) gene” are used interchangeably herein. Acomprehensive review of the Cas protein family is presented in Haft etal. (2005) Computational Biology, PLoS Comput Biol 1 (6): e60.doi:10.1371/journal.pcbi.0010060.

In addition to the four initially described gene families, an additional41 CRISPR-associated (Cas) gene families have been described inWO/2015/026883, which is incorporated herein by reference. Thisreference shows that CRISPR systems belong to different classes, withdifferent repeat patterns, sets of genes, and species ranges. The numberof Cas genes at a given CRISPR locus can vary between species. Casendonuclease relates to a Cas protein encoded by a Cas gene, wherein theCas protein is capable of introducing a double strand break into a DNAtarget sequence. The Cas endonuclease is guided by the guidepolynucleotide to recognize and optionally introduce a double strandbreak at a specific target site into the genome of a cell. As usedherein, the tem “guide polynucleotide/Cas endonuclease system” includesa complex of a Cas endonuclease and a guide polynucleotide that iscapable of introducing a double strand break into a DNA target sequence.The Cas endonuclease unwinds the DNA duplex in close proximity of thegenomic target site and cleaves both DNA strands upon recognition of atarget sequence by a guide nucleotide, but only if the correctprotospacer-adjacent motif (PAM) is approximately oriented at the 3′ endof the target sequence (see FIG. 2A and FIG. 2B of WO/2015/026883,published Feb. 26, 2015).

In an aspect, the Cas endonuclease gene is a Cas9 endonuclease, such as,but not limited to, Cas9 genes listed in SEQ ID NOs: 462, 474, 489, 494,499, 505, and 518 of WO2007/025097, published Mar. 1, 2007, andincorporated herein by reference. In another aspect, the Casendonuclease gene is plant, maize or soybean optimized Cas9endonuclease, such as, but not limited to those shown in FIG. 1A ofWO/2015/026883. In another aspect, the Cas endonuclease gene is operablylinked to a SV40 nuclear targeting signal upstream of the Cas codonregion and a bipartite VirD2 nuclear localization signal (Tinland et al.(1992) Proc. Natl. Acad. Sci. USA 89:7442-6) downstream of the Cas codonregion.

In an aspect, the Cas endonuclease gene is a Cas9 endonuclease gene ofSEQ ID NO:1, 124, 212, 213, 214, 215, 216, 193 or nucleotides 2037-6329of SEQ ID NO:5, or any functional fragment or variant thereof, ofWO/2015/026883.

The terms “functional fragment,” “fragment that is functionallyequivalent,” and “functionally equivalent fragment” are usedinterchangeably herein. These terms refer to a portion or subsequence ofthe Cas endonuclease sequence of the present disclosure in which theability to create a double-strand break is retained.

The terms “functional variant.” “variant that is functionallyequivalent” and “functionally equivalent variant” are usedinterchangeably herein. These terms refer to a variant of the Casendonuclease of the present disclosure in which the ability to create adouble-strand break is retained. Fragments and variants can be obtainedvia methods such as site-directed mutagenesis and syntheticconstruction.

In an aspect, the Cas endonuclease gene is a plant codon optimizedStreptococcus pyogenes Cas9 gene that can recognize any genomic sequenceof the form N(12-30)NGG can in principle be targeted.

Endonucleases are enzymes that cleave the phosphodiester bond within apolynucleotide chain, and include restriction endonucleases that cleaveDNA at specific sites without damaging the bases. Restrictionendonucleases include Type 1, Type II, Type III, and Type IVendonucleases, which further include subtypes. In the Type I and TypeIII systems, both the methylase and restriction activities are containedin a single complex. Endonucleases also include meganucleases, alsoknown as homing endonucleases (HEases), which like restrictionendonucleases, bind and cut at a specific recognition site, however therecognition sites for meganucleases are typically longer, about 18 bp ormore (Patent application PCT/US 12/30061 filed on Mar. 22, 2012).Meganucleases have been classified into four families based on conservedsequence motifs, the families are the LAGLIDADG, GIY-YIG, H-N-H, andHis-Cys box families. These motifs participate in the coordination ofmetal ions and hydrolysis of phosphodiester bonds. Meganucleases arenotable for their long recognition sites, and for tolerating somesequence polymorphisms in their DNA substrates. The naming conventionfor meganuclease is similar to the convention for other restrictionendonuclease. Meganucleases are also characterized by prefix F-, I-, orPI- for enzymes encoded by free-standing ORFs, introns, and inteins,respectively. One step in the recombination process involvespolynucleotide cleavage at or near the recognition site. This cleavingactivity can be used to produce a double-strand break. For reviews ofsite-specific recombinases and their recognition sites, see, Sauer(1994) Curr Op Biotechnol 5:521-7; and Sadowski (1993) FASEB 7:760-7. Insome examples the recombinase is from the Integrase or Resolvasefamilies. TAL effector nucleases are a new class of sequence-specificnucleases that can be used to make double-strand breaks at specifictarget sequences in the genome of a plant or other organism. (Miller, etal. (2011) Nature Biotechnology 29:143-148). Zinc finger nucleases(ZFNs) are engineered double-strand break inducing agents comprised of azinc finger DNA binding domain and a double-strand-break-inducing agentdomain. Recognition site specificity is conferred by the zinc fingerdomain, which typically comprising two, three, or four zinc fingers, forexample having a C2H2 structure, however other zinc finger structuresare known and have been engineered. Zinc finger domains are amenable fordesigning polypeptides which specifically bind a selected polynucleotiderecognition sequence. ZFNs include an engineered DNA-binding zinc fingerdomain linked to a nonspecific endonuclease domain, for example nucleasedomain from a Type Ms endonuclease such as Fok1. Additionalfunctionalities can be fused to the zinc-finger binding domain,including transcriptional activator domains, transcription repressordomains, and methylases. In some examples, dimerization of nucleasedomain is required for cleavage activity. Each zinc finger recognizesthree consecutive base pairs in the target DNA. For example, a 3 fingerdomain recognized a sequence of 9 contiguous nucleotides, with adimerization requirement of the nuclease, two sets of zinc fingertriplets are used to bind an 18 nucleotide recognition sequence.

Bacteria and archaea have evolved adaptive immune defenses termedclustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated (Cas) systems that use short RNA to directdegradation of foreign nucleic acids ((WO2007/025097published Mar. 1,2007). The type II CRISPR/Cas system from bacteria employs a crRNA andtracrRNA to guide the Cas endonuclease to its DNA target. The crRNA(CRISPR RNA) contains the region complementary to one strand of thedouble strand DNA target and base pairs with the tracrRNA(trans-activating CRISPR RNA) forming a RNA duplex that directs the Casendonuclease to cleave the DNA target.

As used herein, the term “guide nucleotide” relates to a syntheticfusion of two RNA molecules, a crRNA (CRISPR RNA) comprising a variabletargeting domain, and a tracrRNA. In an aspect, the guide nucleotidecomprises a variable targeting domain of 12 to 30 nucleotide sequencesand a RNA fragment that can interact with a Cas endonuclease.

As used herein, the term “guide polynucleotide” relates to apolynucleotide sequence that can form a complex with a Cas endonucleaseand enables the Cas endonuclease to recognize and optionally cleave aDNA target site. The guide polynucleotide can be a single molecule or adouble molecule. The guide polynucleotide sequence can be a RNAsequence, a DNA sequence, or a combination thereof (a RNA-DNAcombination sequence). Optionally, the guide polynucleotide can compriseat least one nucleotide, phosphodiester bond or linkage modificationsuch as, but not limited, to Locked Nucleic Acid (LNA), 5-methyl dC,2,6-Diaminopurine, 2′-Fluoro A, 2′-Fluoro U, 2′-O-Methyl RNA,phosphorothioate bond, linkage to a cholesterol molecule, linkage to apolyethylene glycol molecule, linkage to a spacer 18 (hexaethyleneglycol chain) molecule, or 5′ to 3′ covalent linkage resulting incircularization. A guide polynucleotide that solely comprisesribonucleic acids is also referred to as a “guide nucleotide”.

The guide polynucleotide can be a double molecule (also referred to asduplex guide polynucleotide) comprising a first nucleotide sequencedomain (referred to as Variable Targeting domain or VT domain) that iscomplementary to a nucleotide sequence in a target DNA and a secondnucleotide sequence domain (referred to as Cas endonuclease recognitiondomain or CER domain) that interacts with a Cas endonucleasepolypeptide. The CER domain of the double molecule guide polynucleotidecomprises two separate molecules that are hybridized along a region ofcomplementarity. The two separate molecules can be RNA, DNA, and/orRNA-DNA-combination sequences. In an aspect, the first molecule of theduplex guide polynucleotide comprising a VT domain linked to a CERdomain is referred to as “crDNA” (when composed of a contiguous stretchof DNA nucleotides) or “crRNA” (when composed of a contiguous stretch ofRNA nucleotides), or “crDNA-RNA” (when composed of a combination of DNAand RNA nucleotides). The crNucleotide can comprise a fragment of thecRNA naturally occurring in Bacteria and Archaea. In an aspect, the sizeof the fragment of the cRNA naturally occurring in Bacteria and Archaeathat is present in a crNucleotide disclosed herein can range from, butis not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 or more nucleotides.

In an aspect, the second molecule of the duplex guide polynucleotidecomprising a CER domain is referred to as “tracrRNA” (when composed of acontiguous stretch of RNA nucleotides) or “tracrDNA” (when composed of acontiguous stretch of DNA nucleotides) or “tracrDNA-RNA” (when composedof a combination of DNA and RNA nucleotides In an aspect, the RNA thatguides the RNA Cas9 endonuclease complex, is a duplexed RNA comprising aduplex crRNA-tracrRNA.

The guide polynucleotide can also be a single molecule comprising afirst nucleotide sequence domain (referred to as Variable Targetingdomain or VT domain) that is complementary to a nucleotide sequence in atarget DNA and a second nucleotide domain (referred to as Casendonuclease recognition domain or CER domain) that interacts with a Casendonuclease polypeptide. By “domain” it is meant a contiguous stretchof nucleotides that can be RNA, DNA, and/or RNA-DNA-combinationsequence. The VT domain and/or the CER domain of a single guidepolynucleotide can comprise a RNA sequence, a DNA sequence, or aRNA-DNA-combination sequence. In an aspect the single guidepolynucleotide comprises a crNucleotide (comprising a VT domain linkedto a CER domain) linked to a tracrNucleotide (comprising a CER domain),wherein the linkage is a nucleotide sequence comprising a RNA sequence,a DNA sequence, or a RNA-DNA combination sequence. The single guidepolynucleotide being comprised of sequences from the crNucleotide andtracrNucleotide may be referred to as “single guide nucleotide” (whencomposed of a contiguous stretch of RNA nucleotides) or “single guideDNA” (when composed of a contiguous stretch of DNA nucleotides) or“single guide nucleotide-DNA” (when composed of a combination of RNA andDNA nucleotides). In an aspect of the disclosure, the single guidenucleotide comprises a cRNA or cRNA fragment and a tracrRNA or tracrRNAfragment of the type II CRISPR/Cas system that can form a complex with atype II Cas endonuclease, wherein the guide nucleotide Cas endonucleasecomplex can direct the Cas endonuclease to a plant genomic target site,enabling the Cas endonuclease to introduce a double strand break intothe genomic target site. One aspect of using a single guidepolynucleotide versus a duplex guide polynucleotide is that only oneexpression cassette needs to be made to express the single guidepolynucleotide.

The term “variable targeting domain” or “VT domain” is usedinterchangeably herein and includes a nucleotide sequence that iscomplementary to one strand (nucleotide sequence) of a double strand DNAtarget site. The % complementation between the first nucleotide sequencedomain (VT domain) and the target sequence can be at least 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 63%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100%. The variable target domain can beat least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29 or 30 nucleotides in length. In an aspect, the variable targetingdomain comprises a contiguous stretch of 12 to 30 nucleotides. Thevariable targeting domain can be composed of a DNA sequence, a RNAsequence, a modified DNA sequence, a modified RNA sequence, or anycombination thereof.

The term “Cas endonuclease recognition domain” or “CER domain” of aguide polynucleotide is used interchangeably herein and includes anucleotide sequence (such as a second nucleotide sequence domain of aguide polynucleotide), that interacts with a Cas endonucleasepolypeptide. The CER domain can be composed of a DNA sequence, a RNAsequence, a modified DNA sequence, a modified RNA sequence (see forexample modifications described herein), or any combination thereof.

The nucleotide sequence linking the crNucleotide and the tracrNucleotideof a single guide polynucleotide can comprise a RNA sequence, a DNAsequence, or a RNA-DNA combination sequence. In an aspect, thenucleotide sequence linking the crNucleotide and the tracrNucleotide ofa single guide polynucleotide can be at least 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100 nucleotides in length. In another aspect, the nucleotide sequencelinking the crNucleotide and the tracrNucleotide of a single guidepolynucleotide can comprise a tetraloop sequence, such as, but notlimiting to a GAAA tetraloop sequence.

Nucleotide sequence modification of the guide polynucleotide, VT domainand/or CER domain can be selected from, but not limited to, the groupconsisting of a 5′ cap, a 3′ polyadenylated tail, a riboswitch sequence,a stability control sequence, a sequence that forms a dsRNA duplex, amodification or sequence that targets the guide poly nucleotide to asubcellular location, a modification or sequence that provides fortracking, a modification or sequence that provides a binding site forproteins, a Locked Nucleic Acid (LNA), a 5-methyl dC nucleotide, a2,6-Diaminopurine nucleotide, a 2′-Fluoro A nucleotide, a 2′-Fluoro Unucleotide; a 2′-O-Methyl RNA nucleotide, a phosphorothioate bond,linkage to a cholesterol molecule, linkage to a polyethylene glycolmolecule, linkage to a spacer 18 molecule, a 5′ to 3′ covalent linkage,or any combination thereof. These modifications can result in at leastone additional beneficial feature, wherein the additional beneficialfeature is selected from the group of a modified or regulated stability,a subcellular targeting, tracking, a fluorescent label, a binding sitefor a protein or protein complex, modified binding affinity tocomplementary target sequence, modified resistance to cellulardegradation, and increased cellular permeability.

In an aspect, the guide nucleotide and Cas endonuclease are capable offorming a complex that enables the Cas endonuclease to introduce adouble strand break at a DNA target site.

In an aspect of the disclosure the variable target domain is 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30nucleotides in length.

In an aspect of the disclosure, the guide nucleotide comprises a cRNA(or cRNA fragment) and a tracrRNA (or tracrRNA fragment) of the type IICRISPR/Cas system that can form a complex with a type II Casendonuclease, wherein the guide nucleotide Cas endonuclease complex candirect the Cas endonuclease to a plant genomic target site, enabling theCas endonuclease to introduce a double strand break into the genomictarget site. In an aspect the guide nucleotide can be introduced into aplant or plant cell directly using any method known in the art such as,but not limited to, particle bombardment or topical applications.

In an aspect, the guide nucleotide can be introduced indirectly byintroducing a recombinant DNA molecule comprising the correspondingguide DNA sequence operably linked to a plant specific promoter that iscapable of transcribing the guide nucleotide in the plant cell. The term“corresponding guide DNA” includes a DNA molecule that is identical tothe RNA molecule but has a ‘T’ substituted for each “U” of the RNAmolecule.

In an aspect, the guide nucleotide is introduced via particlebombardment or using the disclosed methods and compositions forAgrobacterium transformation of a recombinant DNA construct comprisingthe corresponding guide DNA operably linked to a plant U6 polymerase IIIpromoter.

In an aspect, the RNA that guides the RNA Cas9 endonuclease complex, isa duplexed RNA comprising a duplex crRNA-tracrRNA. One advantage ofusing a guide nucleotide versus a duplexed crRNA-tracrRNA is that onlyone expression cassette needs to be made to express the fused guidenucleotide.

The terms “target site,” “target sequence,” “target DNA,” “targetlocus,” “genomic target site,” “genomic target sequence,” and “genomictarget locus” are used interchangeably herein and refer to apolynucleotide sequence in the genome (including choloroplastic andmitochondrial DNA) of a plant cell at which a double-strand break isinduced in the plant cell genome by a Cas endonuclease. The target sitecan be an endogenous site in the plant genome, or alternatively, thetarget site can be heterologous to the plant and thereby not benaturally occurring in the genome, or the target site can be found in aheterologous genomic location compared to where it occurs in nature.

As used herein, terms “endogenous target sequence” and “native targetsequence” are used interchangeably herein to refer to a target sequencethat is endogenous or native to the genome of a plant and is at theendogenous or native position of that target sequence in the genome ofthe plant. In an aspect, the target site can be similar to a DNArecognition site or target site that that is specifically recognizedand/or bound by a double-strand break inducing agent such as a LIG3-4endonuclease (US patent publication 2009-0133152 A1 (published May 21,2009) or a MS26++ meganuclease (U.S. patent application Ser. No.13/526,912 filed Jun. 19, 2012).

An “artificial target site” or “artificial target sequence” are usedinterchangeably herein and refer to a target sequence that has beenintroduced into the genome of a plant. Such an artificial targetsequence can be identical in sequence to an endogenous or native targetsequence in the genome of a plant but be located in a different position(i.e., a non-endogenous or non-native position) in the genome of aplant.

An “altered target site,” “altered target sequence” “modified targetsite,” and “modified target sequence” are used interchangeably hereinand refer to a target sequence as disclosed herein that comprises atleast one alteration when compared to non-altered target sequence. Such“alterations” include, for example: (i) replacement of at least onenucleotide, (ii) a deletion of at least one nucleotide, (iii) aninsertion of at least one nucleotide, or (iv) any combination of(i)-(iii).

VIII. Methods to Introduce Nucleotides for Site-Specific Integration

In an aspect, the disclosed methods and compositions can be used tointroduce into plants with increased efficiency and speedpolynucleotides useful for the targeted integration of nucleotidesequences into a plant. For example, the disclosed methods andcompositions can be used to introduce transfer cassettes comprisingnucleotide sequences of interest flanked by non-identical recombinationsites are used to transform a plant comprising a target site. In anaspect, the target site contains at least a set of non-identicalrecombination sites corresponding to those on the transfer cassette. Theexchange of the nucleotide sequences flanked by the recombination sitesis affected by a recombinase. Thus, the disclosed methods andcompositions can be used for the introduction of transfer cassettes fortargeted integration of nucleotide sequences, wherein the transfercassettes which are flanked by non-identical recombination sitesrecognized by a recombinase that recognizes and implements recombinationat the nonidentical recombination sites. Accordingly, the disclosedmethods and composition can be used to improve efficiency and speed ofdevelopment of plants, derived from regenerable plant structures,containing non-identical recombination sites.

In an aspect, the present disclosure comprises methods and compositionsfor producing a transgenic plant, wherein the method comprisesintroducing a polynucleotide of interest into a target site in thegenome of a plant cell, the method comprising (a) transforming one ormore cells of an explant with an expression construct comprising: (i) anucleotide sequence encoding a WUS/WOX homeobox polypeptide; (ii) anucleotide sequence encoding a polypeptide comprising two AP2-DNAbinding domains; or (iii) a combination of (i) and (ii); and (b)allowing expression of the polypeptide of (a) in each transformed cellto form a regenerable plant structure in the absence of cytokinin,wherein no callus is formed; wherein transformation further comprisestransforming a cell of an explant with a transfer cassette comprising anucleotide sequence of interest flanked by nonidentical recombinationsites; and wherein the explant is derived from a plant with a genomecomprising a target site flanked by non identical recombination siteswhich correspond to the flanking sites of the transfer cassette. Themethod can further comprise providing a recombinase that recognizes andimplements recombination at the nonidentical recombination sites, therecombinase being provided to the one or more cells of the explant, aregenerable plant structure, a plantlet derived from the regenerableplant structure, or a plant derived from a plantlet derived aregenerable plant structure.

Thus, the disclosed methods and compositions can further comprisecompositions and methods for the directional, targeted integration ofexogenous nucleotides into a transformed plant are provided. In anaspect, the disclosed methods use novel recombination sites in a genetargeting system which facilitates directional targeting of desiredgenes and nucleotide sequences into corresponding recombination sitespreviously introduced into the target plant genome.

In an aspect, a nucleotide sequence flanked by two non-identicalrecombination sites is introduced into one or more cells of an explantderived from the target organism's genome establishing a target site forinsertion of nucleotide sequences of interest. Once a stable plant orcultured tissue is established a second construct, or nucleotidesequence of interest, flanked by corresponding recombination sites asthose flanking the target site, is introduced into the stablytransformed plant or tissues in the presence of a recombinase protein.This process results in exchange of the nucleotide sequences between thenon-identical recombination sites of the target site and the transfercassette.

It is recognized that the transformed plant prepared in this manner maycomprise multiple target sites; i. e., sets of non-identicalrecombination sites. In this manner, multiple manipulations of thetarget site in the transformed plant are available. By target site inthe transformed plant is intended a DNA sequence that has been insertedinto the transformed plant's genome and comprises non-identicalrecombination sites.

Examples of recombination sites for use in the disclosed method areknown in the art and include FRT sites (See, for example, Schlake andBode (1994) Biochemistry 33: 12746-12751; Huang et al. (1991) NucleicAcids Research 19: 443-448; Paul D. Sadowski (1995) In Progress inNucleic Acid Research and Molecular Biology vol. 51, pp. 53-91; MichaelM. Cox (1989) In Mobile DNA, Berg and Howe (eds) American Society ofMicrobiology, Washington D. C., pp. 116-670; Dixon et al. (1995) 18:449-458; Umlauf and Cox (1988) The EMBO Journal 7: 1845-1852; Buchholzet al. (1996) Nucleic Acids Research 24: 3118-3119; Kilby et al. (1993)Trends Genet. 9: 413-421; Rossant and Geagy (1995) Nat. Med. 1: 592-594;Albert et al. (1995) The Plant J. 7: 649-659: Bayley et al. (1992) PlantMol. Biol. 18: 353-361; Odell et al. (1990) Mol. Gen. Genet. 223;369-378; and Dale and Ow (1991) Proc. Natl. Acad. Sci. USA 88:10558-105620; all of which are herein incorporated by reference); Lox(Albert et al. (1995) Plant J. 7: 649-659; Qui et al. (1994) Proc. Natl.Acad. Sci. USA 91: 1706-1710; Stuurman et al. (1996) Plant Mol. Biol.32: 901-913; Odell et al. (1990) Mol. Gen. Gevet. 223: 369-378; Dale etal. (1990) Gene 91: 79-85; and Bayley et al. (1992) Plant Mol. Biol. 18:353-361.) The two-micron plasmid found in most naturally occurringstrains of Saccharomyces cerevisiae, encodes a site-specific recombinasethat promotes an inversion of the DNA between two inverted repeats. Thisinversion plays a central role in plasmid copy-number amplification.

The protein, designated FLP protein, catalyzes site-specificrecombination events. The minimal recombination site (FRT) has beendefined and contains two inverted 13-base pair (bp) repeats surroundingan asymmetric 8-bp spacer. The FLP protein cleaves the site at thejunctions of the repeats and the spacer and is covalently linked to theDNA via a 3′phosphate. Site specific recombinases like FLP cleave andreligate DNA at specific target sequences, resulting in a preciselydefined recombination between two identical sites. To function, thesystem needs the recombination sites and the recombinase. No auxiliaryfactors are needed. Thus, the entire system can be inserted into andfunction in plant cells. The yeast FLP\RT site specific recombinationsystem has been shown to function in plants. To date, the system hasbeen utilized for excision of unwanted DNA. See, Lyznik et at. (1993)Nucleic Acid Res. 21: 969-975. In contrast, the present disclosureutilizes non-identical FRTs for the exchange, targeting, arrangement,insertion and control of expression of nucleotide sequences in the plantgenome.

In an aspect, a transformed organism of interest, such as an explantfrom a plant, containing a target site integrated into its genome isneeded. The target site is characterized by being flanked bynon-identical recombination sites. A targeting cassette is additionallyrequired containing a nucleotide sequence flanked by correspondingnon-identical recombination sites as those sites contained in the targetsite of the transformed organism. A recombinase which recognizes thenon-identical recombination sites and catalyzes site-specificrecombination is required.

It is recognized that the recombinase can be provided by any means knownin the art. That is, it can be provided in the organism or plant cell bytransforming the organism with an expression cassette capable ofexpressing the recombinase in the organism, by transient expression, orby providing messenger RNA (mRNA) for the recombinase or the recombinaseprotein.

By “non-identical recombination sites” it is intended that the flankingrecombination sites are not identical in sequence and will not recombineor recombination between the sites will be minimal. That is, oneflanking recombination site may be a FRT site where the secondrecombination site may be a mutated FRT site. The non-identicalrecombination sites used in the methods of the disclosure prevent orgreatly suppress recombination between the two flanking recombinationsites and excision of the nucleotide sequence contained therein.Accordingly, it is recognized that any suitable non-identicalrecombination sites may be utilized in the disclosure, including FRT andmutant FRT sites, FRT and lox sites, lox and mutant lox sites, as wellas other recombination sites known in the art.

By suitable non-identical recombination site implies that in thepresence of active recombinase, excision of sequences between twonon-identical recombination sites occurs, if at all, with an efficiencyconsiderably lower than the recombinationally-mediated exchangetargeting arrangement of nucleotide sequences into the plant genome.Thus, suitable non-identical sites for use in the disclosure includethose sites where the efficiency of recombination between the sites islow, for example, where the efficiency is less than about 30 to about50%, preferably less than about 10 to about 30%, more preferably lessthan about 5 to about 10%.

As noted above, the recombination sites in the targeting cassettecorrespond to those in the target site of the transformed plant. Thatis, if the target site of the transformed plant contains flankingnon-identical recombination sites of FRT and a mutant FRT, the targetingcassette will contain the same FRT and mutant FRT non-identicalrecombination sites.

It is furthermore recognized that the recombinase, which is used in thedisclosed methods, will depend upon the recombination sites in thetarget site of the transformed plant and the targeting cassette. Thatis, if FRT sites are utilized, the FLP recombinase will be needed. Inthe same manner, where lox sites are utilized, the Cre recombinase isrequired. If the non-identical recombination sites comprise both a FRTand a lox site, both the FLP and Cre recombinase will be required in theplant cell.

The FLP recombinase is a protein which catalyzes a site-specificreaction that is involved in amplifying the copy number of the twomicron plasmid of S. cerevisiae during DNA replication. FLP protein hasbeen cloned and expressed. See, for example, Cox (1993) Proc. Natl.Acad. Sci. U.S.A. 80: 4223-4227. The FLP recombinase for use in thedisclosure may be that derived from the genus Saccharomyces. It may bepreferable to synthesize the recombinase using plant preferred codonsfor optimum expression in a plant of interest. See, for example, U.S.application Ser. No. 08/972,258 filed Nov. 18, 1997, entitled “NovelNucleic Acid Sequence Encoding FLP Recombinase,” herein incorporated byreference.

The bacteriophage recombinase Cre catalyzes site-specific recombinationbetween two lox sites. The Cre recombinase is known in the art. See, forexample, Guo et al. (1997) Nature 389: 40-46; Abremski et al. (1984) J.Biol. Chem. 259: 1509-1514; Chen et al. (1996) Somat. Cell Mol. Genet.22: 477-488; and Shaikh et al. (1977) J. Biol. Chem. 272: 5695-5702. Allof which are herein incorporated by reference. Such Cre sequence mayalso be synthesized using plant preferred codons.

Where appropriate, the nucleotide sequences to be inserted in the plantgenome may be optimized for increased expression in the transformedplant. Where mammalian, yeast, or bacterial genes are used in thedisclosure, they can be synthesized using plant preferred codons forimproved expression. It is recognized that for expression in monocots,dicot genes can also be synthesized using monocot preferred codons.Methods are available in the art for synthesizing plant preferred genes.See, for example, U.S. Pat. Nos. 5,380,831, 5,436,391, and Murray et al.(1989) Nucleic Acids Res. 17: 477-498, herein incorporated by reference.The plant preferred codons may be determined from the codons utilizedmore frequently in the proteins expressed in the plant of interest. Itis recognized that monocot or dicot preferred sequences may beconstructed as well as plant preferred sequences for particular plantspecies. See, for example, EPA 0359472; EPA 0385962; WO 91/16432; Perlaket al. (1991) Proc. Natl. Acad. Sci. USA, 88: 3324-3328; and Murray etal. (1989) Nucleic Acids Research, 17: 477-498. U.S. Pat. Nos.5,380,831; 5,436,391; and the like, herein incorporated by reference. Itis further recognized that all or any part of the gene sequence may beoptimized or synthetic. That is, fully optimized or partially optimizedsequences may also be used.

Additional sequence modifications are known to enhance gene expressionin a cellular host and can be used in the disclosure. These includeelimination of sequences encoding spurious polyadenylation signals,exon-intron splice site signals, transposon-like repeats, and other suchwell-characterized sequences, which may be deleterious to geneexpression. The G-C content of the sequence may be adjusted to levelsaverage for a given cellular host, as calculated by reference to knowngenes expressed in the host cell. When possible, the sequence ismodified to avoid predicted hairpin secondary RNA structures.

The present disclosure also encompasses novel FLP recombination targetsites (FRT). The FRT has been identified as a minimal sequencecomprising two 13 base pair repeats, separated by an 8 base spacer, asfollows: 5′-GAAGTTCCTATTC [TCTAGAAA] GTATAGGAACTTC3′ wherein thenucleotides within the brackets indicate the spacer region. Thenucleotides in the spacer region can be replaced with a combination ofnucleotides, so long as the two 13-base repeats are separated by eightnucleotides. It appears that the actual nucleotide sequence of thespacer is not critical; however for the practice of the disclosure, somesubstitutions of nucleotides in the space region may work better thanothers. The eight base pair spacer is involved in DNA-DNA pairing duringstrand exchange. The asymmetry of the region determines the direction ofsite alignment in the recombination event, which will subsequently leadto either inversion or excision. As indicated above, most of the spacercan be mutated without a loss of function. See, for example, Schlake andBode (1994) Biochemistry 33: 12746-12751, herein incorporated byreference.

Novel FRT mutant sites can be used in the practice of the disclosedmethods. Such mutant sites may be constructed by PCR-based mutagenesis.Although mutant FRT sites are known (see SEQ ID Nos 2, 3, 4 and 5 ofWO/1999/025821, published May 27, 1999), it is recognized that othermutant FRT sites may be used in the practice of the disclosure. Thepresent disclosure is not the use of a particular FRT or recombinationsite, but rather that non-identical recombination sites or FRT sites canbe utilized for targeted insertion and expression of nucleotidesequences in a plant genome. Thus, other mutant FRT sites can beconstructed and utilized based upon the present disclosure.

As discussed above, bringing genomic DNA containing a target site withnon-identical recombination sites together with a vector containing atransfer cassette with corresponding non-identical recombination sites,in the presence of the recombinase, results in recombination. Thenucleotide sequence of the transfer cassette located between theflanking recombination sites is exchanged with the nucleotide sequenceof the target site located between the flanking recombination sites. Inthis manner, nucleotide sequences of interest may be preciselyincorporated into the genome of the host.

It is recognized that many variations of the disclosure can bepracticed. For example, target sites can be constructed having multiplenon-identical recombination sites. Thus, multiple genes or nucleotidesequences can be stacked or ordered at precise locations in the plantgenome. Likewise, once a target site has been established within thegenome, additional recombination sites may be introduced byincorporating such sites within the nucleotide sequence of the transfercassette and the transfer of the sites to the target sequence. Thus,once a target site has been established, it is possible to subsequentlyadd sites, or alter sites through recombination.

Another variation includes providing a promoter or transcriptioninitiation region operably linked with the target site in an organism.Preferably, the promoter will be 5′ to the first recombination site. Bytransforming the organism with a transfer cassette comprising a codingregion, expression of the coding region will occur upon integration ofthe transfer cassette into the target site. This aspect provides for amethod to select transformed cells, particularly plant cells, byproviding a selectable marker sequence as the coding sequence.

Other advantages of the present system include the ability to reduce thecomplexity of integration of transgenes or transferred DNA in anorganism by utilizing transfer cassettes as discussed above andselecting organisms with simple integration patterns. In the samemanner, preferred sites within the genome can be identified by comparingseveral transformation events. A preferred site within the genomeincludes one that does not disrupt expression of essential sequences andprovides for adequate expression of the transgene sequence.

The disclosed methods also provide for means to combine multiplecassettes at one location within the genome. Recombination sites may beadded or deleted at target sites within the genome.

Any means known in the art for bringing the three components of thesystem together may be used in the disclosure. For example, a plant canbe stably transformed to harbor the target site in its genome. Therecombinase may be transiently expressed or provided. Alternatively, anucleotide sequence capable of expressing the recombinase may be stablyintegrated into the genome of the plant. In the presence of thecorresponding target site and the recombinase, the transfer cassette,flanked by corresponding non-identical recombination sites, is insertedinto the transformed plant's genome.

Alternatively, the components of the system may be brought together bysexually crossing transformed plants. In this aspect, a transformedplant, parent one, containing a target site integrated in its genome canbe sexually crossed with a second plant, parent two, that has beengenetically transformed with a transfer cassette containing flankingnon-identical recombination sites, which correspond to those in plantone. Either plant one or plant two contains within its genome anucleotide sequence expressing recombinase. The recombinase may be underthe control of a constitutive or inducible promoter.

Inducible promoters include those described herein above, as well as,heat-inducible promoters, estradiol-responsive promoters, chemicalinducible promoters, and the like. Pathogen inducible promoters includethose from pathogenesis-related proteins (PR proteins), which areinduced following infection by a pathogen; e. g., PR proteins, SARproteins, beta-1,3-glucanase, chitinase, etc. See, for example, Redolfiet al. (1983) Neth. J. Plant Pathol. 89: 245-254; Uknes et al. (1992)The Plant Cell 4: 645-656; and Van Loon (1985) Plant Mol. Virol. 4:111-116. In this manner, expression of recombinase and subsequentactivity at the recombination sites can be controlled.

Constitutive promoters for use in expression of genes in plants areknown in the art. Such promoters include, but are not limited to 35Spromoter of cauliflower mosaic virus (Depicker et al. (1982) Mol. Appl.Genet. 1: 561-573; Odell et al. (1985) Nature 313: 810-812), ubiquitinpromoter (Christensen et al. (1992) Plant Mol. Biol. 18: 675-689),promoters from genes such as ribulose bisphosphate carboxylase (DeAlmeida et al. (1989) Mol. Gen. Genet. 218; 78-98), actin (McElroy etal. (1990) Plant J. 2: 163-171), histone, DnaJ (Baszczynski et al.(1997) Maydica 42: 189-201), and the like.

The disclosed compositions and methods are useful in targeting theintegration of transferred nucleotide sequences to a specificchromosomal site. The nucleotide sequence may encode any nucleotidesequence of interest. Particular genes of interest include those whichprovide a readily analyzable functional feature to the host cell and/ororganism, such as marker genes, as well as other genes that alter thephenotype of the recipient cells, and the like. Thus, genes effectingplant growth, height, susceptibility to disease, insects, nutritionalvalue, and the like may be utilized in the disclosure. The nucleotidesequence also may encode an ‘antisense’ sequence to turn off or modifygene expression.

It is recognized that the nucleotide sequences will be utilized in afunctional expression unit or cassette. By functional expression unit orcassette is intended, the nucleotide sequence of interest with afunctional promoter, and in most instances a termination region. Thereare various ways to achieve the functional expression unit within thepractice of the disclosure. In one aspect of the disclosure, the nucleicacid of interest is transferred or inserted into the genome as afunctional expression unit.

Alternatively, the nucleotide sequence may be inserted into a sitewithin the genome which is 3′ to a promoter region. In this latterinstance, the insertion of the coding sequence 3′ to the promoter regionis such that a functional expression unit is achieved upon integration.For convenience, for expression in plants, the nucleic acid encodingtarget sites and the transfer cassettes, including the nucleotidesequences of interest, can be contained within expression cassettes. Theexpression cassette will comprise a transcriptional initiation region,or promoter, operably linked to the nucleic acid encoding the peptide ofinterest. Such an expression cassette is provided with a plurality ofrestriction sites for insertion of the gene or genes of interest to beunder the transcriptional regulation of the regulatory regions.

The transcriptional initiation region, the promoter, may be native orhomologous or foreign or heterologous to the host, or could be thenatural sequence or a synthetic sequence. By foreign is intended thatthe transcriptional initiation region is not found in the wild-type hostinto which the transcriptional initiation region is introduced. Either anative or heterologous promoter may be used with respect to the codingsequence of interest.

The transcriptional cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region, aDNA sequence of interest, and a transcriptional and translationaltermination region functional in plants. The termination region may benative with the transcriptional initiation region, may be native withthe DNA sequence of interest, or may be derived from another source.Convenient termination regions are available from the potato proteinaseinhibitor (PinII) gene or sequences from Ti-plasmid of A. tumefaciens,such as the nopaline synthase, octopine synthase and opaline synthasetermination regions. See also, Guerineau et al., (1991) Mol. Gen. Genet.262: 141-144; Proudfoot (1991) Cell 64: 671-674; Sanfacon et al. (1991)Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272;Munroe et al. (1990) Gene 91: 151-158; Ballas et al. 1989) Nucleic AcidsRes. 17: 7891-7903; Joshi et al. (1987) Nucleic Acid Res. 15: 9627-9639.

The expression cassettes may additionally contain 5′ leader sequences inthe expression cassette construct. Such leader sequences can act toenhance translation. Translation leaders are known in the art andinclude: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′noncoding region) (Elroy-Stein, O., Fuerst, T.R., and Moss, B. (1989) PNAS USA, 86: 6126-6130); potyvirus leaders, forexample, TEV leader (Tobacco Etch Virus)(Allison et al. (1986); MDMVleader (Maize Dwarf Mosaic Virus); Virology, 154: 9-20), and humanimmunoglobulin heavy-chain binding protein (BiP), (Macejak, D. G., andP. Sarnow (1991) Nature, 353: 90-94; untranslated leader from the coatprotein MARNA of alfalfa mosaic virus (AMV RNA 4), (Jobling, S. A., andGehrke, L., (1987) Nature, 325: 622-625; tobacco mosaic virus leader(TMV), (Gallie et al. (1989) Molecular Biology of RNA, pages 237-256,Gallie et al. (1987) Nucl. Acids Res. 15: 3257-3273; maize chloroticmottle virus leader (MCMV) (Lornmel, S. A. et al. (1991) Virology, 81:382-385). See also, Della-Cioppa et al. (1987) Plant Physiology, 84:965-968; and endogenous maize 5′ untranslated sequences. Other methodsknown to enhance translation can also be utilized, for example, introns,and the like.

The expression cassettes may contain one or more than one gene ornucleic acid sequence to be transferred and expressed in the transformedplant. Thus, each nucleic acid sequence will be operably linked to 5′and 3′ regulatory sequences. Alternatively, multiple expressioncassettes may be provided.

EXPERIMENTAL Example 1: Plasmids

Plasmids comprising T-DNA described in Table 1 were used in experimentsdescribed herein below. The listed plasmids in Table 1 harbor a T-DNAcontaining the indicated components.

TABLE 1 Plasmid Components. Plasmid ID T-DNA PHP77833 RB + NOSPRO:Top2:ZM-WUS2::IN2-1 TERM + ZM-PLTP PRO::ZM-ODP2::OS-T28 TERM +GZ-W64A TERM + UBI PRO:UBI1ZM INTRON:ESR::SB-SAG12 TERM + SB-ALS PRO::HRA::SB-PEPC1 TERM + LTP2 PRO::ZS- YELLOW::PINII TERM-LB (SEQ ID NO:22). PHP78156 RB + NOS PRO:Top2:WUS2::IN2-1 TERM + DR5PRO:Top3:ODP2::OS-T28 TERM + GZ-W64A TERM + UBI PRO::ESR::SB-SAG12TERM + SB-ALS PRO:: HRA::SB- PEPC1 TERM + LTP2 PRO::ZS-YELLOW::PINIITERM-LB (SEQ ID NO: 24). PHP78157 RB + NOS PRO:Top2:ZM-WUS2::IN2-1TERM + ZM-PLTP PRO:Top:ZM-ODP2::OS-T28 TERM + GZ-W64A TERM + UBIPRO:UBI1ZM INTRON:ESR::SB-SAG12 TERM + SB-ALS PRO:: HRA::SB-PEPC1 TERM +LTP2 PRO::ZS- YELLOW::PINII TERM-LB (SEQ ID NO: 23). PHP79023 RB + DR5PRO::ZM-WUS2::PINII TERM + ZM-PLTP PRO::ZM-ODP2::OS-T28 TERM + GZ-W64ATERM + UBI PRO:UBI1ZM INTRON:ESR::SB-SAG12 TERM + SB-ALS PRO::HRA::SB-PEPC1 TERM + UBI PRO:UBI1ZM INTRON:ZS-GREEN1::PINIITERM:SB-ACTIN TERM-LB (SEQ ID NO: 25). PHP79024 RB + ZM-AXIG1PRO:Top1:ZM-WUS2::IN2-1 TERM + ZM- PLTP PRO::ZM-ODP2::OS-T28 TERM +GZ-W64A TERM + UBI PRO:UBI1ZM INTRON:ESR::SB-SAG12 TERM + SB-ALS PRO::HRA::SB-PEPC1 TERM + UBI PRO::ZS-GREEN1::PINII TERM:SB-ACTIN TERM-LB(SEQ ID NO: 26). PHP79066 RB-ZM-AXIG1 PRO:Top1:ZM-WUS2::In2-1 TERM + ZM-PLTP PRO::ZM-ODP2::OS-T28 TERM + SB-ALS PRO::HRA::SB-PEPC1 TERM + LTP2PRO::ZS-YELLOW N1::PINII TERM-LB (SEQ ID NO: 28). PHP80334 RB +LOXP-ZM-AXIG1 PRO:Top1:ZM-WUS2::IN2-1 TERM + ZM-PLTP PRO:ZM-ODP2::OS-T28TERM:PINII TERM:CZ19B1 TERM + UBI PRO:UBI1ZM INTRON:MO-CRE EχON1:ST-LS1INTRON2:MO-CRE EXON2::PINII + SB-UBI PRO::ZS-GREEN1::PINII TERM:SB-ACTINTERM + CAMV35S PRO::ADH1 INTRON1::ESR::SB-SAG12 TERM- LOXP + SB-ALSPRO:: HRA::PINII TERM-LB (SEQ ID NO: 41) PHP80338 RB + LOXP-ZM-AXIG1PRO:Top1:WUS2::IN2-1 TERM + ZM- PLTP PRO:ZM-ODP2::OS-T28 TERM:PINIITERM:CZ19B1 TERM + GLB1 PRO::MO-CRE EXON1:ST-LS1 INTRON2:MO- CREEXON2::PINII + SB-UBI PRO::ZS-GREEN1::PINII TERM:SB-ACTIN TERM + CAMV35SPRO::ADH1 INTRON1::ESR::SB-SAG12 TERM-LOXP + SB-ALS PRO:: HRA::PINIITERM-LB (SEQ ID NO: 42) PHP38332 RB + UBI PRO:UBI1 ZM INTRON:PMI::PINIITERM + UBI PRO:UBI1 ZM INTRON:MO-PAT::ZS-YELLOW-N1::PINII TERM -LB (SEQID NO: 43). PHP80921 RB + SI-UBI3 PRO::SI-UBI3 INTRON1::ZS-GREEN1::PINIITERM + SB-ALS PRO:HRA:SB-PEPC1 TERM + LB (SEQ ID NO: 44). PHP80560 RB +LOXP-ZM-AXIG1 PRO::ZM-WUS2::IN2-1 TERM + ZM- PLTP PRO::ZM-ODP2::OS-T28TERM::PINII TERM::CZ19B1 TERM + ZM-IN2-2 PRO::MO-CRE EXON1:ST-LS1INTRON2::MO-CRE EXON2::PINII + SB-UBI PRO::ZS- GREEN1::PINIITERM::SB-ACTIN TERM + LOXP + SB-ALS PRO:: HRA::PINII TERM-LB (SEQ ID NO:44). PHP82240 RB + LOXP-ZM-AXIG1 PRO::Top1::WUS2::IN2-1 TERM + ZM-PLTPPRO:ZM-ODP2::OS-T28 TERM::PINII TERM:CZ19B1 TERM + ZM-GOS2 PRO::SB-UBIINTRON1::EC-LEXA-ZM-CI::SB-ACTIN TERM + 6X EC- RECA::CAMV 35S-MINPRO::DS-RED::OS-UBI TERM + SB- ALS PRO:: HRA::PINII TERM-LB (SEQ ID NO:85). PHP81814 RB + SB-ALS PRO::HRA EXON1:: LOXP + ZM-AXIG1PRO::ZM-WUS2::IN2-1 TERM + ZM-PLTP PRO::ZM- ODP2::OS-T28 TERM::PINIITERM::CZ19B1 TERM + ZM- GLB1 PRO::MO-CRE EXON1:ST-LS1 INTRON2::MO-CREEXON2::PINII + SB-UBI PRO::ZS-GREEN1::OS-UBI TERM + LOXP-HRAEXON2::SB-PEPC1 TERM + -LB (SEQ ID NO: 80). PHP80558 RB + LOXP-ZM-AXIG1PRO::Top1::WUS2::IN2-1 TERM + ZM-PLTP PRO:ZM-ODP2::OS-T28 TERM:PINIITERM::CZ19B1 TERM + ZM-WOX2A PRO::MO-CRE EXON1:ST-LS1 INTRON2::MO-CREEXON2::PINII + SB-UBI PRO::ZS-GREEN1::PINII TERM::OS-UBI TERM + LOXP +SB-ALS PRO:: HRA::PINII TERM-LB (SEQ ID NO: 74) PHP24600 RB + CAMV35STERM:: PAT:: CAMVS PRO + UBIZM PRO:: DS-RED:: PINII TERM + LB (SEQ IDNO: 69) PHP79530 RB + LOXP + NOS PRO::OS-WUS::IN2-1 TERM + SB-UBIPRO::SB-UBI-INTRON1::OS-ODP2::SB-UBI TERM + GZ- W64A TERM + UBI1ZMPRO::UBI1ZM INTRON1::ZS- GREEN1::PINII TERM + SB-ACTIN TERM + LOXP +SB-ALS PRO::ZM-ALS::PINII TERM + LB (SEQ ID NO: 70) PHP79531 RB + LOXP +NOS PRO::SI-WUS::IN2-1 TERM + SB-UBIPRO::SB-UBI-INTRON1::SI-ODP2::SB-UBI TERM + GZ- W64A TERM + UBI1ZMPRO::UBI1ZM INTRON1::ZS- GREEN1::PINII TERM + SB-ACTIN TERM + LOXP +SB-ALS PRO::ZM-ALS::PINII TERM + LB (SEQ ID NO: 71) PHP80911 RB + LOXP +NOS PRO::ZM-WUS2::IN2-1 TERM + SB-UBIPRO::SB-UBI-INTRON1::ZM-ODP2::SB-UBI TERM + GZ- W64A TERM + UBI1ZMPRO::UBI1ZM INTRON1::ZS- GREEN1::PINII TERM + SB-ACTIN TERM + LOXP +SB-ALS PRO::ZM-ALS::PINII TERM + LB (SEQ ID NO: 72) PHP80912 RB +ZM-PLTP PRO::ZM-ODP2::OS-T28 TERM + SB-ALS PRO::ZM-ALS::SB-PEPC1 TERM +LTP2 PRO::ZS- YELLOWN1::PINII TERM + LB (SEQ ID NO: 53) PHP80913 RB +ZM-AXIG1 PRO::ZM-WUS2::IN2-1 TERM + SB-ALS PRO::ZM-ALS::SB-PEPC1 TERM +LTP2 PRO::ZS- YELLOWN1::PINII TERM + LB (SEQ ID NO: 54) RV003866 RB- UBIPRO:UBI1ZM INTRON::MO-FLP::PINII TERM + CaMV35S TERM + FRT1::PMI::PINIITERM + ZM-AXIG1 PRO::ZM-WUS2::IN2-1 TERM + ZM-PLTP PRO::ZM- ODP2::OS-T28TERM + UBI PRO::UBI1ZM INTRON::DsRED: FRT87-LB (SEQ ID NO: 89) RV004886RB- + ZM-AXIG1 PRO::ZM-WUS2::IN2-1 TERM + ZM-PLTP PRO::ZM-ODP2::OS-T28TERM + UBI PRO:: UBI1ZM INTRON::MO-FLP::PINII TERM + CaMV35S TERM +FRT1::PMI::PINII TERM + UBI PRO::UBI1ZM INTRON::DsRED: FRT87-LB (SEQ IDNO: 90) RV012587 RB + ZM-PLTP PRO::ZM-LEC1::IN2-1 TERM + ZM-PLTPPRO::ZM-ODP2:: OS-T28 TERM + SB-UBI PRO:SB-UBIINTRON1::ZS-GREEN1::OS-UBI TERM + SB-ALS PRO:: HRA::PINII TERM + LB (SEQID NO: 91) RV012588 RB + ZM-AXIG1 PRO::ZM-WUS2::IN2-1 TERM + ZM-PLTPPRO::ZM-LEC1:: OS-T28 TERM + SB-UBI PRO::SB-UBIINTRON1::ZS-GREEN1::OS-UBI TERM + SB-ALS PRO:: HRA::PINII TERM + LB (SEQID NO: 92) RV012589 RB + ZM-CAB7 PRO::ZM-WUS2::IN2-1 TERM + ZM-PLTPPRO::ZM-ODP2:: OS-T28 TERM + SB-UBI PRO::SB-UBIINTRON1::ZS-GREEN1::OS-UBI TERM + SB-ALS PRO:: HRA::PINII TERM + LB (SEQID NO: 93) RV012590 RB + ZM-UVBR PRO::ZM-WUS2::IN2-1 TERM + ZM-PLTPPRO::ZM-ODP2:: OS-T28 TERM + SB-UBI PRO::SB-UBIINTRON1::ZS-GREEN1::OS-UBI TERM + SB-ALS PRO:: HRA::PINII TERM + LB (SEQID NO: 94) RV012591 RB + ZM-AXIG1 PRO::ZM-WUS2::IN2-1 TERM + SB-PLTP1PRO::ZM-ODP2:: OS-T28 TERM + SB-UBI PRO:SB-UBIINTRON1::ZS-GREEN1::OS-UBI TERM + SB-ALS PRO:: HRA::PINII TERM + LB (SEQID NO: 95) RV012592 RB + ZM-AXIG1 PRO::ZM-WUS2::IN2-1 TERM + SI-PLTP1PRO::ZM-ODP2:: OS-T28 TERM + SB-UBI PRO::SB-UBIINTRON1::ZS-GREEN1::OS-UBI TERM + SB-ALS PRO:: HRA::PINII TERM + LB (SEQID NO: 96) RV012593 RB + ZM-AXIG1 PRO::ZM-WUS2::IN2-1 TERM + ZM-PLTP1PRO::ZM-ODP2:: OS-T28 TERM + SB-UBI PRO:SB-UBIINTRON1::ZS-GREEN1::OS-UBI TERM + SB-ALS PRO:: HRA::PINII TERM + LB (SEQID NO: 97) RV012594 RB + ZM-AXIG1 PRO::ZM-WUS2::IN2-1 TERM + ZM-PLTP2PRO::ZM-ODP2:: OS-T28 TERM + SB-UBI PRO::SB-UBIINTRON1::ZS-GREEN1::OS-UBI TERM + SB-ALS PRO:: HRA::PINII TERM + LB (SEQID NO: 98) RV012595 RB + ZM-AXIG1 PRO::ZM-WUS2::IN2-1 TERM + OS-PLTP1PRO::ZM-ODP2:: OS-T28 TERM + SB-UBI PRO:SB-UBIINTRON1::ZS-GREEN1::OS-UBI TERM + SB-ALS PRO:: HRA::PINII TERM + LB (SEQID NO: 99) RV012603 RB + ZM-AXIG1 PRO::ZM-WOX2A::IN2-1 TERM + ZM-PLTPPRO::ZM-ODP2:: OS-T28 TERM + SB-UBI PRO:SB-UBIINTRON1::ZS-GREEN1::OS-UBI TERM + SB-ALS PRO:: HRA::PINII TERM + LB (SEQID NO: 100) RV012604 RB + ZM-AXIG1 PRO::ZM-WOX4::IN2-1 TERM + ZM-PLTPPRO::ZM-ODP2:: OS-T28 TERM + SB-UBI PRO:SB-UBI INTRON1:ZS-GREEN1::OS-UBITERM + SB-ALS PRO:: HRA::PINII TERM + LB (SEQ ID NO: 101) RV012605 RB +ZM-AXIG1 PRO::ZM-WOX5A::IN2-1 TERM + ZM-PLTP PRO::ZM-ODP2:: OS-T28TERM + SB-UBI PRO::SB-UBI INTRON1::ZS-GREEN1::OS-UBI TERM + SB-ALS PRO::HRA::PINII TERM + LB (SEQ ID NO: 102) RV012606 RB + ZM-AXIG1PRO::SB-WUS1::IN2-1 TERM + ZM-PLTP PRO::ZM-ODP2:: OS-T28 TERM + SB-UBIPRO:SB-UBI INTRON1::ZS-GREEN1::OS-UBI TERM + SB-ALS PRO:: HRA::PINIITERM + LB (SEQ ID NO: 103) RV012608 RB + ZM-AXIG1 PRO::ZM-WUS2::IN2-1TERM + ZM-PLTP PRO::ZM-ODP2:: OS-T28 TERM + SB-UBI PRO::SB-UBIINTRON1::ZS-GREEN1::OS-UBI TERM + SB-ALS PRO:: HRA::PINII TERM + LB (SEQID NO: 104) PHP80730 OVERDRIVE + RB (OCTOPINE) + GM-LTP3 PRO::AT-WUS::UBQ14 TERM + GM-UBQ PRO::GM-UBQ INTRON1::TAG-RFP::UBQ3 TERM +GM-SAMS PRO::GM- SAMS INTRON1::GM-HRA::GM-ALS TERM + LB (OCTOPINE) + LB(AGROPINE) + LB (SEQ ID NO: 105)

Example 2: Culture Media

Various media are referenced in the Examples for use in transformationand cell culture. Media compositions are described below in Tables 2-9.

TABLE 2 Culture media for sorghum transformation. Medium CompositionPHI-I: 4.3 g/l MS salts (Phytotechnology Laboratories, Shawnee Mission,KS, catalog number M524), 0.5 mg/l nicotinic acid, 0.5 mg/l pyridoxineHCl, 1 mg/l thiamine HCl, 0.1 g/l myo-inositol, 1 g/l casamino acids(Becton Dickinson and Company, BD Diagnostic Systems, Sparks, MD,catalog number 223050), 1.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D),68.5 g/l sucrose, 36 g/l glucose, pH 5.2; with 100 μM acetosyringoneadded before using. PHI-T: PHI-I with 20 g/l sucrose, 10 g/l glucose, 2mg/l 2,4-D, no casamino acids, 0.5 g/l MES buffer, 0.7 g/l L-proline, 10mg/l ascorbic acid, 100 μM acetosyringone, 8 g/l agar, pH 5.8. PHI-U:PHI-T with 1.5 mg/l 2,4-D 100 mg/l carbenicillin, 30 g/l sucrose, noglucose and acetosyringone; 5 mg/l PPT, pH 5.8. PHI-UM: PHI-U with12.5g/l mannose and 5 g/l maltose, no sucrose, no PPT, pH 5.8 PHI-V: PHI-Uwith 10 mg/l PPT DBC3: 4.3 g/l MS salts, 0.25 g/l myo-inositol, 1.0 g/lcasein hydrolysate, 1.0 mg/l thiamine HCL, 1.0 mg/l 2,4-D, 30 g/lmaltose, 0.69 g/l L-proline, 1.22 mg/l cupric sulfate, 0.5 mg/l BAP(6-benzylaminopurine), 3.5 g/l phytagel, pH 5.8 PHI-X: 4.3 g/l MS salts,0.1 g/l myo-inositol, 5.0 ml MS vitamins stock^(b), 0.5 mg/l zeatin, 700mg/l L-proline, 60 g/l sucrose, 1 mg/l indole-3-acetic acid, 0.1 μMabscisic acid, 0.1 mg/l thidiazuron, 100 mg/l carbenicillin, 5 mg/l PPT,8 g/l agar, pH 5.6. PHI-XM: PHI-X with no PPT; added 1.25 mg/l cupricsulfate, pH 5.6. PHI-Z: 2.15 g/l MS salts, 0.05 g/l myo-inositol, 2.5 mlMS vitamins stock^(b), 20 g/l sucrose, 3 g/l phytagel, pH 5.6 ^(a)PHI-I,PHI-T, PHI-U, PHI-V, PHI-X, and PHI-Z media from Zhao et al. 2000 ^(b)MSvitamins stock: 0.1 g/l nicotinic acid, 0.1 g/l pyridoxine HCl, 0.02 g/lthiamine HCl, 0.4 g/l glycine.

TABLE 3 Composition of wheat liquid infection medium WI 4. WI 4 DI water1000 mL MS salt + Vitamins(M519) 4.43 g Maltose 30 g Glucose 10 g MES1.95 g 2,4-D (.5 mg/L) 1 ml Picloram (10 mg/ml) 200 μl BAP (1 mg/L) .5ml Adjust PH to 5.8 with KOH Post sterilization add: Acetosyringone (400μM) 400 μl

TABLE 4 Composition of wheat co-cultivation medium WC#10. WC # 10 DIwater 1000 mL MS salt + Vitamins(M519) 4.43 g Maltose 30 g Glucose 1 gMES 1.95 g 2,4-D (.5 mg/L) 1 ml Picloram (10 mg/ml) 200 μl BAP (1 mg/L).5 ml 50X CuSO4 (.1M) 49 μl Adjust PH to 5.8 with KOH and add 2.5 g/L ofPhytagel. Post sterilization add: Acetosyringone (400 μM) 400 μl

TABLE 5 Composition of wheat Green Tissue culture medium DBC4. DBC4 ddH20 1000 mL MS salt 4.3 g Maltose 30 g Myo-inositol 0.25 g N-Z-Amine-A 1g Proline 0.69 g Thiamine-HCl (0.1 mg/mL) 10 mL 50X CuSO4 (0.1M) 49 μL2,4-D (0.5 mg/mL) 2 mL BAP 1 mL Adjust PH to 5.8 with KOH and then add3.5 g/L of Phytagel. Post sterilization add: Cef(100 mg/ml) 1 ml

TABLE 6 Composition of wheat Green Tissue induction medium DBC6. DBC6 ddH20 1000 mL MS salt 4.3 g Maltose 30 g Myo-inositol 0.25 g N-Z-Amine-A 1g Proline 0.69 g Thiamine-HCl (0.1 mg/mL) 10 mL 50X CuSO4 (0.1M) 49 μL2,4-D (0.5 mg/mL) 1 mL BAP 2 mL Adjust PH to 5.8 with KOH and then add3.5 g/L of Phytagel. Post sterilization add: Cef(100 mg/ml) 1 ml

TABLE 7 Composition of wheat regeneration medium MSA. MSA dd H20 1000 mLMS salt + Vitamins(M519) 4.43 g Sucrose 20 g Myo- Inositol 1 g Adjust PHto 5.8 with KOH and then add 3.5 g/L of Phytagel. Post steriliaztionadd: Cef(100 mg/ml) 1 ml

TABLE 8 Composition of wheat regeneration medium MSB. MSB dd H20 1000 mLMS salt + Vitamins(M519) 4.43 g Sucrose 20 g Myo- Inositol 1 g Adjust PHto 5.8 with KOH and then add 3.5 g/L of Phytagel. Post steriliaztionadd: Cef(100 mg/ml) 1 ml IBA .5 ml

TABLE 9 Media formations for maize transformation, selection andregeneration. Units Medium components per liter 12V 810I 700 710I 605J605T 289Q MS BASAL SALT g 4.3 4.3 4.3 4.3 4.3 MIXTURE N6 MACRONUTRIENTSml 60.0 60.0 10X POTASSIUM NITRATE g 1.7 1.7 B5H MINOR SALTS ml 0.6 0.61000X NaFe EDTA FOR B5H ml 6.0 6.0 100X ERIKSSON'S ml 0.4 0.4 VITAMINS1000X S&H VITAMIN STOCK ml 6.0 6.0 100X THIAMINE•HCL mg 10.0 10.0 0.50.5 L-PROLINE g 0.7 2.0 2.0 0.7 CASEIN g 0.3 0.3 HYDROLYSATE (ACID)SUCROSE g 68.5 20.0 20.0 20.0 60.0 GLUCOSE g 5.0 36.0 10.0 0.6 0.6MALTOSE g 2,4-D mg 1.5 2.0 0.8 0.8 AGAR g 15.0 15.0 8.0 6.0 6.0 8.0PHYTAGEL g DICAMBA g 1.2 1.2 SILVER NITRATE mg 3.4 3.4 AGRIBIOCarbenicillin mg 100.0 Timentin mg 150.0 150.0 Cefotaxime mg 100.0 100.0MYO-INOSITOL g 0.1 0.1 0.1 NICOTINIC ACID mg 0.5 0.5 PYRIDOXINE•HCL mg0.5 0.5 VITAMIN ASSAY g 1.0 CASAMINO ACIDS MES BUFFER g 0.5ACETOSYRINGONE uM 100.0 ASCORBIC ACID mg 10.0 10 MG/ML (7S) MS VITAMINSTOCK ml 5.0 SOL. ZEATIN mg 0.5 CUPRIC SULFATE mg 1.3 IAA 0.5 MG/ML(28A) ml 2.0 ABA 0.1 mm ml 1.0 THIDIAZURON mg 0.1 AGRIBIO Carbenicillinmg 100.0 PPT(GLUFOSINATE- mg NH4) BAP mg 1.0 YEAST EXTRACT (BD g 5.0Difco) PEPTONE g 10.0 SODIUM CHLORIDE g 5.0 SPECTINOMYCIN mg 50.0 100.0FERROUS ml 2.0 SULFATE•7H20 AB BUFFER 20X (12D) ml 50.0 AB SALTS 20X(12E) ml 50.0 Benomyl mg pH 5.6 Units Medium components per liter 289R13158H 13224B 13266K 272X 272V 13158 MS BASAL SALT g 4.3 4.3 4.3 4.3 4.34.3 MIXTURE N6 MACRONUTRIENTS ml 4.0 60.0 10X POTASSIUM NITRATE g 1.7B5H MINOR SALTS ml 0.6 1000X NaFe EDTA FOR B5H ml 6.0 100X ERIKSSON'S ml1.0 0.4 VITAMINS 1000X S&H VITAMIN STOCK ml 6.0 100X THIAMINE•HCL mg 0.50.5 L-PROLINE g 0.7 0.7 2.9 2.0 CASEIN g 0.3 HYDROLYSATE (ACID) SUCROSEg 60.0 60.0 190.0 20.0 40.0 40.0 40.0 GLUCOSE g 0.6 MALTOSE g 2,4-D mg1.6 AGAR g 8.0 6.4 6.0 6.0 6.0 6.0 PHYTAGEL g DICAMBA g 1.2 SILVERNITRATE mg 8.5 1.7 AGRIBIO Carbenicillin mg 2.0 Timentin mg 150.0 150.0Cefotaxime mg 100.0 100.0 25 25 MYO-INOSITOL g 0.1 0.1 0.1 0.1 0.1NICOTINIC ACID mg PYRIDOXINE•HCL mg VITAMIN ASSAY g CASAMINO ACIDS MESBUFFER g ACETOSYRINGONE uM ASCORBIC ACID mg 10 MG/ML (7S) MS VITAMINSTOCK ml 5.0 5.0 5.0 5.0 5.0 SOL. ZEATIN mg 0.5 0.5 CUPRIC SULFATE mg1.3 1.3 IAA 0.5 MG/ML (28A) ml 2.0 2.0 ABA 0.1 mm ml 1.0 1.0 THIDIAZURONmg 0.1 0.1 AGRIBIO Carbenicillin mg PPT(GLUFOSINATE- mg NH4) BAP mgYEAST EXTRACT (BD g Difco) PEPTONE g SODIUM CHLORIDE g SPECTINOMYCIN mgFERROUS ml SULFATE•7H20 AB BUFFER 20X (12D) ml AB SALTS 20X (12E) mlBenomyl mg 100.0 pH 0.5 5.6

Example 3: Particle Bombardment

Prior to bombardment, 10-12 DAP immature embryos were isolated from earsof the Pioneer inbred PH184C and placed on culture medium plus 16%sucrose for three hours to plasmolyze the scutellar cells.

Four plasmids were typically used for each particle bombardment; 1) thedonor plasmid (100 ng/μl) containing the FRT-flanked donor cassette forRecombinase-Mediated Cassette Exchange, 2) a plasmid (2.5 ng/μl)containing the expression cassette UBI PRO::FLPm::PinII, 3) a plasmid(10 ng/μl) containing the expression cassette UBI PRO::ODP2::PinII, and4) a plasmid (5 ng/ul) containing the expression cassetteUBI::WUS2::PinII. To attach the DNA to 0.6 μm gold particles, the fourplasmids were mixed by adding 10 μl of each plasmid together in alow-binding microfuge tube (Sorenson Bioscience 39640T) for a total of40 μl. To this suspension, 50 μl of 0.6 μm gold particles (30 μg/μl) and1.0 μl of Transit 20/20 (Cat No MIR5404, Mirus Bio LLC) were added, andthe suspension was placed on a rotary shaker for 10 minutes. Thesuspension was centrifuged at 10,000 RPM (˜9400×g) and the supernatantwas discarded. The gold particles were re-suspended in 120 μl of 100%ethanol, briefly sonicated at low power and 10 μl was pipetted onto eachflier. The fliers were then air-dried to remove all the remainingethanol. Particle bombardment was performed using a Biolistics PDF-1000,at 28 inches of Mercury using a 200 PSI rupture disc.

Example 4: Agrobacterium-Mediated Transformation of Corn A. Preparationof Agrobacterium Master Plate.

Agrobacterium tumefaciens harboring a binary donor vector was streakedout from a −80° C. frozen aliquot onto solid 12V medium and cultured at28° C. in the dark for 2-3 days to make a master plate.

B. Growing Agrobacterium on Solid Medium.

A single colony or multiple colonies of Agrobacterium were picked fromthe master plate and streaked onto a second plate containing 8101 mediumand incubated at 28° C. in the dark for 1-2 days. Agrobacteriuminfection medium (700 medium; 5 ml) and 100 mM3′-5′-Dimethoxy-4′-hydroxyacetophenone (acetosyringone; 5 μL) were addedto a 14 mL conical tube in a hood. About 3 full loops of Agrobacteriumfrom the second plate were suspended in the tube and the tube was thenvortexed to make an even suspension. Suspension (1 ml) was transferredto a spectrophotometer tube and the optical density (550 nm) of thesuspension was adjusted to a reading of about 0.35-2.0. TheAgrobacterium concentration was approximately 0.5 to 2.0×10⁹ cfu/mL. Thefinal Agrobacterium suspension was aliquoted into 2 mL microcentrifugetubes, each containing about 1 mL of the suspension. The suspensionswere then used as soon as possible.

C. Growing Agrobacterium on Liquid Medium.

Alternatively, Agrobacterium can be prepared for transformation bygrowing in liquid medium. One day before infection, a 125 ml flask wasprepared with 30 ml of 557A medium (10.5 g/l potassium phosphatedibasic, 4.5 g/l potassium phosphate monobasic anhydrous, 1 g/l ammoniumsulfate, 0.5 g/l sodium citrate dehydrate, 10 g/l sucrose, 1 mMmagnesium sulfate) and 30 μL spectinomycin (50 mg/mL) and 30 μLacetosyringone (20 mg/mL). A half loopful of Agrobacterium from a secondplate was suspended into the flasks and placed on an orbital shaker setat 200 rpm and incubated at the 28° C. overnight. The Agrobacteriumculture was centrifuged at 5000 rpm for 10 min. The supernatant wasremoved and the Agrobacterium infection medium with acetosyringonesolution was added. The bacteria were resuspended by vortex and theoptical density (550 nm) of Agrobacterium suspension was adjusted to areading of about 0.35 to 2.0.

D. Maize Transformation.

Ears of a maize (Zea mays L.) cultivar were surface-sterilized for 15-20min in 20% (v/v) bleach (5.25% sodium hypochlorite) plus 1 drop of Tween20 followed by 3 washes in sterile water. Immature embryos (IEs) wereisolated from ears and were placed in 2 ml of the Agrobacteriuminfection medium with acetosyringone solution. The optimal size of theembryos varies based on the inbred, but for transformation with WUS2 andODP2 a wide size range of immature embryo sizes could be used. Thesolution was drawn off and 1 ml of Agrobacterium suspension was added tothe embryos and the tube vortexed for 5-10 sec. The microfuge tube wasallowed to stand for 5 min in the hood. The suspension of Agrobacteriumand embryos were poured onto 710I co-cultivation medium (see Table 9).Any embryos left in the tube were transferred to the plate using asterile spatula. The Agrobacterium suspension was drawn off and theembryos placed axis side down on the media. The plate was sealed withParafilm M® film (moisture resistant flexible plastic, available atBemis Company, Inc., 1 Neenah Center 4^(th) floor, PO Box 669, Neenah,Wis. 54957) and incubated in the dark at 21° C. for 1-3 days ofco-cultivation.

Embryos were transferred to resting medium (605T medium) withoutselection. Three to 7 days later, they were transferred to maturationmedium (289Q medium) supplemented with a selective agent.

Example 5: Expression of ODP2 and WUS2

The following experiment demonstrated that expression of ODP2 and WUS2immediately after Agrobacterium infection resulted in direct somaticembryogenesis.

A. The PLTP promoter driving ODP2 and the NOS promoter driving WUS2expression resulted in rapid, direct somatic embryo formation.

Immature embryos (2-2.5 mm in length) were harvested from Pioneer maizeinbred PH184C approximately 11 days after pollination, and were infectedwith Agrobacterium strain AGL1 containing a T-DNA with the followingcomposition; RB+NOS PRO::Top2::ZM-WUS2::IN2-1 TERM+ZM-PLTPPRO::ZM-ODP2::OS-T28 TERM+GZ-W64A TERM+UBI PRO::UBI1ZMINTRON::ESR::SB-SAG12 TERM+SB-ALS PRO:: HRA::SB-PEPC1 TERM+LTP2PRO::ZS-YELLOW::PINII TERM-LB, as described in Example 3. For the PLTPPRO, see SEQ ID NO. 1. Agrobacterium was grown in liquid medium to anoptical density of 0.5 (at 520 nm) and the immature embryos (53, 52 and56 embryos from three separate ears) were incubated in the Agrobacteriumsuspension for 5 minutes before removal from the liquid to be placed onsolid 710I medium.

After 24 hours, the embryos were moved to 605T medium to begin selectionagainst the Agrobacterium. After 6 days, numerous small somatic embryoswere observed on the surface of each of the 124 treated immatureembryos. Each immature embryo contained numerous, distinct, individualsomatic embryos, many being supported on clearly-defined suspensors.Representative embryos produced by expression of ODP2 and WUS 2 areshown in FIG. 1 and FIG. 2, e.g., see arrows pointing to newly formedembryos in FIG. 1. Newly-formed fluorescent embryos are also seen inFIG. 2. The image in FIG. 1 was captured 4 days after the beginning ofAgrobacterium infection, using a stereomicroscope with lighting fromabove. The overall length of the zygotic embryo is approximately 1.5 mm.The embryos shown in FIG. 2 were transformed with the AXIG1::WUS2:: IN2and PLTP::ODP2::OS-T28 expression cassettes, along with a UBIPRO::ZS-GREEN::PNII expression cassette. The image shows fluorescentembryos growing on the scutellar surface of the originally-transformedzygotic embryo following treatment using the disclosed methods. Thisimage was captured 4 days after the beginning of Agrobacteriuminfection, using a stereomicroscope with epifluorecence attachments anda standard Leica GFP filter set. The overall length of the zygoticembryo is approximately 1.5 mm.

Seven days after Agro-infection, the embryos were transferred tomaturation medium (289Q medium with 0.1 mg/I imazapyr), usingimidazolinone herbicide to select for transgenic embryos. After 14 dayson the maturation medium, the mature embryos were moved onto rootingmedium (13158H medium; 13158 medium plus 25 mg/i cefotaxime) and leafpieces were sampled for PCR analysis. From the 53 embryos derived fromthe first ear, 12 herbicide-resistant plants were analyzed using PCRanalysis and sent to the greenhouse between 32-34 days after thebeginning of the experiment, which was begun when the Agrobacteriumtransformation was started. Plants were sampled for PCR by taking twosamples from each plant, one from each of two opposing leaves (fromopposite sides of the plant) to confirm that the plants were notchimeric. PCR results for each pair of samples from all the plantsindicated that no chimeric plants were produced, and that the T0 plantswere homogenously transgenic.

B. Comparing no herbicide selection, no herbicide selection with theaddition of branched chain amino acids, or selection with imazapyr.

Two replicates of this experiment were performed, with the onlydifferences between the two replicates were the number of startingimmature embryos and the numbers of embryos (or plants) moving on tosuccessive stages of the experiment. For both replicates, immatureembryos from the Pioneer inbred PH184C were infected with Agrobacteriumstrain AGL1 (THY-) containing the following T-DNA (from PHP77833);RB+NOS PRO::Top2::ZM-WUS2::IN2-1 TERM+ZM-PLTP PRO::ZM-ODP2::OS-T28TERM+GZ-W64A TERM+UBI PRO:UBI1ZM INTRON:ESR::SB-SAG12 TERM+SB-ALS PRO::HRA::SB-PEPC1 TERM+LTP2 PRO::ZS-YELLOW::PINII TERM-LB. After a 5 minuteinfection in the liquid Agrobacterium suspension, the immature embryoswere transferred to solid culture medium (2701 media) overnight. Thefollowing day the embryos were moved onto one of three media, 605T; 605Twith 0.1 mg/l ethametsulfuron (also containing the herbicide imazapyr(0.1 mg/l) for selection) plus branched chain amino acids (abbreviatedas “AA”, containing 100 mM leucine, isoleucine, or valine); or 605T with0.1 mg/l ethametsulfuron (also containing the herbicide imazapyr (0.1mg/l) for selection) with no branched-chain amino acids. Embryos weremoved to maturation medium 12 days later, with embryos from 605T mediumbeing transferred to 289Q medium with 0.1 mg/l imazapyr (selectionduring maturation), embryos from medium 605T medium with 0.1 mg/lethametsulfuron with AA being transferred to 289Q medium (earlyselection but no selection during maturation), and embryos from medium605T with 0.1 mg/I ethametsulfuron moving onto 289Q medium (earlyselection). Sixteen days later, embryos with healthy somatic embryoswere moved onto regeneration medium 272V medium.

For the first replicate of this experiment, 206 embryos were treatedwith Agrobacterium and one day later 106, 67, or 33 embryos were movedonto 605T medium (no selection for the first week), 605T medium with 0.1mg/I ethametsulfuron with AA (early selection with AA) or 605T mediumwith 0.1 mg/l ethametsulfuron (early selection with no AA),respectively. For the next transfer, 45, 16 and 0 embryos were movedonto their respective maturation media. For the final transfer torooting medium, 18, 10 and 0 plantlets (individual events) were moved.Of these events, 16 and 10 were sampled for PCR and 6 and 2 plants werefound to be single copy for the integrated transgenes, with 0 and 2escapes (wild-type plants that survived the selection process) wereobserved, respectively. For this replicate of the experiment, the totalelapsed time from Agrobacterium infection to the greenhouse was 48 days.These results indicated that early selection with no continued selectioninto embryo maturation allowed wild-type escapes to survive (along withsome transgenic events). However, early selection without supplementalbranched-chain amino acids appeared to be stressful during the stagewhen the somatic embryos were forming and thus no events were produced.Starting selection at the beginning of the maturation stage was the mosteffective for event recovery with no escapes.

For the second replicate, a total of 196 embryos were treated withAgrobacterium in liquid for 5 minutes and then co-cultured for one dayon 710I medium. At this point, 94, 66 or 36 embryos were moved onto 605Tmedium, 605T medium with 0.1 mg/l ethametsulfuron with AA or 605T mediumwith 0.1 mg/I ethametsulfuron, respectively. Twelve days later, the 94embryos on 605T were split (47 each) onto either 289Q medium with 0.1mg/l imazapyr or onto 289Q medium with 0.5 mg/l imazapyr. The embryosfrom both the 605T medium with 0.1 mg/l ethametsulfuron with AA and 605Tmedium with 0.1 mg/l ethametsulfuron were moved onto 289Q (no furtherselection). After maturation, healthy plantlets (events) weretransferred to rooting medium 13158H, with 14, 11, 13 and 0 events beingmoved from the above four maturation treatments, respectively.Ultimately, 4, 5 and 2 plants were sent to the greenhouse for the threetreatments from which events were recovered, and only one single-copyevent was produced from the 0.5 mg/l imazapyr selection duringmaturation.

C. Comparing two promoters, ZM-PLTP PRO::Top1 and the DR5 PRO::Top3,driving expression of ODP2.

For both treatments, immature embryos from Pioneer inbred PH184C wereharvested, treated with Agrobacterium strain AGL1 (THY-) containing therespective plasmids, were transferred onto 605T medium after one day onco-cultivation medium 710I with the Agrobacterium, were cultured on 605Tmedium for 10 days, moved onto 13226D with 0.1 mg/l ethametsulfuron fortwo days and then transferred onto maturation medium (289Q with 0.1 mg/Iimazapyr) for 13 days. After maturation, the plantlets (events) weremoved onto rooting (germination) medium 13158 for ten days (rootingstage).

i. ZM-PLTP Promoter Driving ZM-ODP2 Expression.

Eighty immature embryos were treated with Agrobacterium containingPHP78157 (SEQ ID NO. 23). Initially, all treated immature embryosresponded by rapidly producing many individual somatic embryos on thesurface of each scutellum. At the end of the rooting stage, 18 plantletswere produced. A subset of ten plants were sent to the greenhouse andsampled for PCR analysis (total elapsed time to the greenhouse was 46days). Of these ten, six were multi-copy and/or contained plasmidbackbone (BB), and there were 4 escapes.

ii. DR5 Promoter Driving ODP2 Expression.

Seventy immature embryos were treated with Agrobacterium containingPHP78156, harboring the T-DNA containing the following: RB+NOSPRO::Top2::WUS2::IN2-1 TERM+DR5 PRO:Top3:ODP2::OS-T28 TERM+GZ-W64ATERM+UBI PRO::ESR::SB-SAG12 TERM+SB-ALS PRO:: HRA::SB-PEPC1 TERM+LTP2PRO::ZS-YELLOW::PINII TERM-LB (SEQ ID NO: 24).

Initially, all treated immature embryos responded by rapidly producingmany individual somatic embryos on the surface of each scutellum. At theend of the rooting stage, 18 plantlets were produced. A subset of 12plants were sent to the greenhouse and sampled for PCR analysis (totalelapsed time to the greenhouse was 46 days). Of these twelve plantsanalyzed by PCR, 6 were multi-copy/BB+, 4 were single-copy andbackbone-free (BB−), and there were 2 escapes.

Example 6: ZM-PLTP::ZM-ODP2+ZM-AXIG1 PRO::ZM-WUS2

The expression of ODP2 under the control of the PLTP promoter and theexpression of WUS2 under the control of the AXIG1 promoter resulted inimproved plant recovery, high frequencies of plants sent to thegreenhouse and higher frequency of single-copy events.

Immature embryos were harvested from two maize inbreds (HC69 and PH184C)and were infected with Agrobacterium (strain LBA4404 THY-) containingeither PHP79023 (SEQ ID NO.: 25) or PHP79024 (SEQ ID NO.: 26). Forinbred HC69, 67 immature embryos were transformed with PHP79024,ultimately producing 26 plants that were resistant to 0.1 mg/A imazapyr(38.8% frequency relative to the number of starting embryos). Bycomparison, when 65 HC69 embryos were transformed with PHP79023, 10herbicide-resistant plants were sent to the greenhouse (15.4%frequency). In addition, the plants produced using PHP79024 were morevigorous and healthier in the greenhouse. Transgenic plants from bothstarting plasmids were sent to the greenhouse 33 days after infectionwith Agrobacterium.

For inbred PH184C, 64 immature embryos were transformed with PHP79024,ultimately producing 30 plants that were resistant to 0.1 mg/I imazapyr(46.8% frequency relative to the number of starting embryos). Bycomparison, when 73 PH184Cembryos were transformed with PHP79023, 27herbicide-resistant plants were sent to the greenhouse (37% frequency).In addition, the plants produced using PHP79024 were more vigorous andhealthier in the greenhouse. Transgenic plants from both startingplasmids were sent to the greenhouse 33 days after infection withAgrobacterium. Thus for both inbreds, using the DR5 promoter to driveexpression of WUS2 (along with PLTP PRO::ODP2) rapidly producedtransgenic somatic embryos that could be germinated into transgenic T0plants, but the combination of AXIG1 PRO::WUS2+PLTP PRO::ODP2 was evenmore effective, both in terms of the overall frequency of transgenicplant production but also in terms of T0 vigor and health in thegreenhouse.

Example 7: Separation of Somatic Embryos from the Supporting Scutellum

The recovery of independent transgenic plants was improved by separationof somatic embryos from the supporting scutellum of the original zygoticimmature embryos.

Immature embryos from Pioneer inbred PHH5G were transformed usingAgrobacterium strain AGL1 (THY-) harboring the plasmid PHP79066 (SEQ IDNO.: 28) (see Table 1). After co-cultivation of 45 immature embryos withAgrobacterium on medium 710I for one day, the immature embryos weremoved onto resting medium (605T medium) with no selection for 12 days.The embryos were moved onto maturation medium for 13 days, and multiplemature somatic embryos were clearly observed as being derived from asingle original zygotic immature embryo (FIG. 3). At this point, theembryos were removed from solid medium and placed in a liquid culture(289R medium). The tube containing the suspended corn tissue was thenvortexed on high power for 30-60 seconds, visually inspecting thesuspension every 10-15 seconds until it appeared that the tissue wasdispersed. The liquid suspension was then poured onto solid rootingmedium (13158H medium with 0.1 mg/l imazapyr herbicide) and the liquidwas pipetted off the plate. From this material, over 200 plants wereproduced (representative plants shown in FIG. 4), which resulted in anaverage of 4.4 plants being recovered for every starting embryo that wastransformed. Of the approximately 200 T0 plants, 152 were sampled forPCR analysis, all were transformed based on PCR results (no escapes) and43 plants were single copy for the transgenes with no Agro backbone(28.3% single-copy no BB). Based on this frequency, the total number ofrecovered T0 plants and the starting number of embryos, the productionof quality, usable T0 events (single-copy, no backbone) was over 100%based on the number of starting embryos.

Example 8: Cre-Mediated Excision A. UBI::CRE::PINII-Mediated Excision.

Immature embryos were harvested from Pioneer inbreds HC69 (293 embryos)and PH184C (241 embryos), and were transformed with Agrobacterium strainAGL1 (THY-) harboring the plasmid PHP80334 (SEQ ID NO.: 42) containingthe genetic elements shown in Table 1. After 5 minutes exposure to theAgrobacterium in liquid medium 710I, the embryos were plated out ontoagar-solidified 710I medium for overnight co-cultivation. After one dayof co-cultivation, embryos were moved onto resting medium (605T with noselection) for 7 days, transferred to maturation medium (289Q with 0.1mg/l imazapyr) for 14 days, and then were moved onto rooting(germination) medium for at least 14 days before moving the plants tothe greenhouse. Based on the original number of embryos transformed,19.1% and 13.8% of the original embryos produced a transgenic plant inHC69 and PH184C, respectively. To determine the excision frequency,seven PCR reactions were performed to test for the absence of the CRE,WUS2, ODP2, ZS-GREEN1 and ESR expression cassettes (i.e., allpolynucleotide segments that were originally between the two LOXP sitesin the T-DNA) and the presence of the HRA expression cassette. PCRresults revealed that 21% of the HC69 plants contained only the HRAexpression cassette (conferring resistance to the herbicide imazapyr)with the segment between the two original LOXP sites having excised.Thus, these plants no longer contained the CRE, WUS2, ODP2, ZS-GREEN1AND ESR expression cassettes. When plants were analyzed for the presenceof the HRA cassette with all the other cassettes having been excised,the excision frequency for PH184C was even higher than for HC69, with47% of the plants showing that complete, perfect excision had occurred.

A difference was observed in the early growth response when the plasmidcontaining UBI PRO::CRE was compared to constructs that were notdesigned for excision. In contrast to non-excision constructs containingAXIG1 PRO::WUS2+PLTP PRO::ODP2, wherein somatic embryos were visible at6 days after Agrobacterium infection, there were no fluorescent somaticembryos observed in the UBI PRO::CRE treatment during this interval. Theubiquitin promoter (UBI PRO) is a very strong, constitutive promoterthat has been used widely over many years (Christensen and Quail, (1996)Transgenic Research 5:213-218). Due to its strength and ubiquitousexpression pattern, it was anticipated that UBI PRO::CRE would beginexpressing immediately upon being introduced into the cell, thus it wasanticipated that the WUS and ODP2 expression cassettes could be excisedbefore being able to stimulate somatic embryo formation. Surprisingly,however, when the original zygotic embryos were placed on maturationmedium, it was observed that multiple embryos formed on what hadoriginally been a single zygotic embryo. Maturation on the herbicideimazapyr was a very stringent selection against wild-type growth, andconsistent with this, the PCR results confirmed the resultant plantswere transgenic and that 60% of the single-copy T0 plants analyzed byPCR showed the occurrence of perfect excision of WUS, ODP2, CRE,ZS-GREEN1 and ESR The T-DNA of PHP80334 contains the AXIG1 PRO::WUS2,PLTP PRO::ODP2, UBI PRO::CRE, UBI PRO::ZS-GREEN and ESR expressioncassettes located within the loxP recombination sites. When this T-DNAwas introduced into immature embryo cells, no green fluorescence wasobserved indicating that excision occurred before green fluorescenceprotein could accumulate to visible levels (typically less than 24 hoursin a non-excised UBI PRO::ZS-GREEN expression cassette. These data showthat a short pulse of WUS and ODP2 are effective for rapid de novosomatic embryo formation and germination of these somatic embryos toproduce transgenic T0 plants.

B. GLB1::CRE-Mediated Excision.

Immature embryos were harvested from Pioneer inbreds PH184C. and weretransformed with Agrobacterium strain AGL1 (THY-) harboring the plasmidPHP80338 (SEQ ID NO.: 43) containing the genetic elements shown inTable 1. Transformation, selection and plant germination methods werethe same as those described for the UBI PRO::CRE experiment in Example8-A above. PCR analysis of T0 plants demonstrated that 58% of thesingle-copy plants had undergone complete excision of the developmentalgenes (ODP2 and WUS) and CRE. Substituting promoters known to driveexpression either during embryo development (LTP2, OLE, END-2) or beingstimulated by stress (IN2-1) also resulted in excision of thedevelopmental genes (ODP2, WUS2) and CRE before germination to producetransgenic T0 plants.

In a comparison of different promoters driving expression of CRE,immature embryos were harvested from either Pioneer inbred HC69 orPH184C and were transformed with Agrobacterium-strain LBA4404 (Thy-)harboring a plasmid containing a T-DNA with the following components;RB-loxP-AXIG1 PRO::WUS2::In2-1 TERM+PLTP PRO::ODP2::PINII TERM+“XPRO”::CRE::OS-128 TERM-loxP+SB-ALS PRO::ZM-HRA::PINII TERM, where “XPRO” was the maize UBI PRO, the WOX2a PRO, the IN2 PRO, the LTP2 PRO,the OLE PRO, the GLB1 PRO, the HSP17.7 PRO or the HSP26 PRO that wereused to drive excision of WUS, ODP2 and CRE. After 5 minutes exposure tothe Agrobacterium in liquid medium 710I, the embryos were plated outonto agar-solidified 710I medium for overnight co-cultivation. After theone day co-cultivation, embryos were moved onto resting medium (605Twith no selection) for 7 days, transferred to maturation medium with(289Q with 0.1 mg/l imazapyr) for 14 days, and then were moved ontorooting (germination) medium for at least 14 days before moving theplants to the greenhouse.

TABLE 10 Transformation and excision frequencies for promoters drivingexpression of CRE recombinase. Exci- Promoter No. No. sion driving CREof Em- of TXN Fre- Inbred Vector Recombinase bryos Plants % quencyPH184C PHP80558 WOX2a 140 56 40%  0% PH184C PHP80560 IN2 173 34 20%  0%PH184C PHP80334 UBI 440 40  9% 60% PH184C PHP80559 LTP2 229 75 33% 50%PH184C PHP80561 OLE 228 59 26% 40% PH184C PHP80770 GLB1 327 72 22% 47%HC69 PHP81430 HSP17.7 190 102 54% 67% HC69 PHP81431 HSP26 242 78 32% 54%

As shown in Table 10, transformation efficiencies were measured by thenumber of recovered T0 plants from a given number of starting immatureembryos and excision frequencies are shown for WUS2. ODP2 and CRE. Table10 also shows the number of immature embryos that were infected withAgrobacterium, the number of T0 plants produced, the calculatedtransformation frequency [(No. of T0 plants/No. of embryosinfected)*100] and the final excision frequency based on PCR analysisfor the presence or absence of WUS, ODP2, CRE and HRA. For inbredPH184C, no excision was observed in the negative control treatments inwhich the T-DNA contained either WOX2a::CRE::PINII or IN2::CRE::PINII(PHP80558 or PHP80560, respectively). However, for treatments in whichthe promoter driving CRE was strongly expressed in the whole plant(UBI), were expressed strongly in developing embryos (LTP2, OLE, GLB1)or were strongly induced by heat treatment (HSP17.7, HSP26), excisionfrequencies ranged between 40 and 60%. For the UBI PRO, excision of WUS2and ODP2 was rapid resulting in a reduced transformation frequency (9%)and the excision frequency as measured in the T0 plants was high at 60%.For the promoters expressed during mid- to late-embryo development(LTP2, OLE and GLB1) the transformation frequencies were higher than forUBI (33, 26 and 22%, respectively) while the excision frequencies wereslightly lower than for UBI (50.40 and 47%, respectively). The treatmentin which the HSP17.7 was used to control CRE expression produced theoverall best results in both categories, producing the highesttransformation frequency (54%) and the highest excision frequency (67%).HSP26 produced results that were in the same range as theembryo-promoters, with transformation and excision frequencies of 32%and 54%, respectively.

When the embryo-developmental promoters were used for excision, numeroussomatic embryos were observed in these treatments (similar toexperiments where no excision components were present) which indicatedthat expression was delayed until a later stage of embryo developmentbut when developmentally-triggered, expression was uniformly strongthroughout the embryos resulting in efficient excision of WUS2, ODP2 andCRE. For the heat-responsive promoters, exposure to heat (i.e. 42° C.for two hours repeated on three consecutive days) was deferred until theembryos were moved onto maturation medium, resulting in high numbers ofsomatic embryos that formed rapidly after transformation, followed bylater efficient excision.

Example 9: Improved Recovery of Site-Specific Recombination Events

Immature ears were harvested from PH184C and 2.0 mm immature embryoswere extracted from the kernels on the day of the particle bombardmenttreatment. The embryos were placed on high osmotic medium (13224Bmedium) for three hours prior to particle bombardment. Immature embryoswere bombarded with an equimolar ratio of plasmids containing thefollowing expression cassettes; FRT1:PMI::PINII TERM:FRT87+UBIPRO:UBI1ZM INTRON::MO-FLP::PINII TERM+ZM-PLTP PRO::ZM-ODP2::PINIITERM+ZM-AXIG1 PRO::ZM-WUS2::PINII TERM. After particle bombardment, theimmature embryos remained on the high-osmotic medium overnight, and werethen transferred to resting medium (13266K medium with 150 mg/l G418)for 8 days. After the resting period, the embryos were transferred tomaturation medium (289O medium with 150 mg/l G418) for 21 days, and thenmoved onto rooting medium (272X medium with 150 mg/l G418) for 14-17days (until the roots were large enough for transplanting into soil). Atthe plantlet stage, leaf tissue was sampled for PCR analysis to confirmthat the genes within the flanking FRT1 and FRT87 sites of the originaltarget locus were no longer present and that the new genes within thedonor cassette had recombined into the target locus correctly—andprecise RMCE (Recombinase-Mediated Cassette Exchange) events wereidentified. This reduced the entire SSI cycle, from transformation tohaving precise RMCE-derived plants in the greenhouse, down to 43-50days, depending on how long a time was required to produce adequateroots. Alternately, an Agrobacterium mediated SSI method was developedusing constructs with AXIG1 PRO::WUS2+PLTP PTO::ODP2. Two T-DNAs weredelivered in two separate experiments; the first T-DNA containing PMI,WUS2, ODP2 and DsRED expression cassettes within the flanking FRT1 andFRT87 recombination sites (RB− UBI PRO:UBI1ZM INTRON::MO-FLP::PINIITERM+CaMV35S TERM+FRT1:PMI::PINII TERM+ZM-AXIG1 PRO::ZM-WUS2::IN2-1TERM+ZM-PLTP PRO::ZM-ODP2::OS-T28 TERM+UBI PRO::UBI1ZM INTRON::DsRED:FRT87-LB) (RV003866) and the second T-DNA containing only PMI and DsREDwithin the FRT1 and FRT87 sties (RB−+ZM-AXIG1 PRO::ZM-WUS2::IN2-1TERM+ZM-PLTP PRO::ZM-ODP2::OS-T28 TERM UBI PRO:UBI1ZMINTRON::MO-FLP::PINII TERM+CaMV35S TERM+FRT1:PMI::PINII TERM+UBIPRO::UBI1ZM INTRON::DsRED: FRT87-LB) (RV004886). Each of the T-DNAs weredelivered via Agro-mediated transformation into target lines withFRT1-FRT87 landing sites as described in U.S. Provisional Appln. No.62/296,639, herein incorporated in entirety by reference. Precise RMCEevents were identified using a multiplex PCR assay as described in #5907USPSP and the data is summarized in Table 11. The use of AXIG1PRO::WUS2+PLTP PTO::ODP2 expression cassettes for Agro SSI reduced theentire SSI process by several weeks (at least 3-4 weeks), compared tothe normal transformation method for generating SSI events.

TABLE 11 Recovery of RMCE events in Agro SSI experiments using WUS2 andODP2 expression. # embryos # T0 # % Vector infected plants RMCE RMCERV003866 859 42 6 0.7 RV004886 836 10 1 0.1

Example 10: Improved Recovery of Events Containing CAS9/Crispr-MediatedGenomic Modifications

CAS9-mediated cutting of the maize genome is used to introduce singlecodon changes to the maize ALS2 gene. To generate ALS2 edited alleles, a794 bp fragment of homology is cloned into a plasmid vector and two 127nucleotide single-stranded DNA oligos are tested as repair template,containing several nucleotide changes in comparison to the nativesequence. The 794 bp repair templates include a single nucleotide changewhich will direct editing of DNA sequences corresponding to the prolineat amino acid position 165 changing to a serine (P165S), as well asthree additional changes within the ALS-CR4 target site and PAMsequence. Modification of the PAM sequence within the repair templatealters the methionine codon (AUG) to isoleucine (AUU), which naturallyoccurs in the ALS1 gene. The media used for particle bombardment,selection and regeneration are similar to those used herein.Approximately 1,000 immature embryos per treatment are bombarded withthe two oligo or single plasmid repair templates, UBI PRO:UBI1ZMINTRON:CAS9::PINII, POLIII PRO::ALS-CR4 gRNA, UBI PRO:UBI1ZMINTRON:MOPAT˜DSRED::PINII TERM, ZM-PLTP PRO::ZM-ODP2::PINII TERM andZM-AXIG1 PRO::ZM-WUS2::PINII TERM. After particle bombardment, theimmature embryos were placed on media containing 3 mg/l bialaphos toselect for herbicide-resistant somatic embryos. After the resting periodof 8 days, the embryos were transferred onto maturation medium withbiolaphos for 21 days, and then are moved onto rooting medium for 14-17days (until the roots were large enough for transplanting into soil).Five weeks post-transformation, two hundred (per treatment) randomlyselected independent young plantlets growing on selective media weretransferred to fresh bialaphos media in PlantCon™ containers (sterileplastic containers that can accommodate plants up to 6″ in height). Theremaining plantlets (approximately 800 per treatment) were transferredto the solid media within PlantCons™ (presterilized plant tissue culturecontainer available from MP Biomedicals LLC, 3 Hutton Center Drive,Suite 100. Santa Anna, Calif. 92707) containing 100 ppm ofchlorosulfuron as direct selection for an edited ALS2 gene. One monthlater, 384 of the randomly sampled plantlets (with no selection), andseven plantlets that survived chlorsulfuron selection were sampled foranalysis. Edited ALS2 alleles were detected in 9 plantlets: two derivedfrom the randomly-selected plantlets growing on bialaphos and generatedusing the 794 bp repair DNA template, and the remaining 7 derived fromchlorosulfuron resistant plantlets edited using the 127 ntsingle-stranded oligos. Analysis of the ALS1 gene revealed onlywild-type sequence confirming high specificity of the ALS-CR4 gRNA.

All nine plants containing edited ALS2 alleles were sent to thegreenhouse and sampled for additional molecular analysis and progenytesting. DNA sequence analysis of ALS2 alleles confirms the presence ofthe P165S modification as well as the other nucleotide changesassociated with the respective repair templates. T1 and T2 progeny oftwo T0 plants were analyzed to evaluate the inheritance of the editedALS2 alleles. Progeny plants derived from crosses using pollen from wildtype Hi-II plants were analyzed by sequencing and demonstrated sexualtransmission of the edited alleles observed in the parent plant withexpected 1:1 segregation ratio (57:56 and 47:49, respectively). To testwhether the edited ALS sequence conferred herbicide resistance, selectedfour-week old segregating T1 plants with edited and wild-type ALS2alleles were sprayed with four different concentrations of chlorsulfuron(50, 100 (Ox), 200, and 400 mg/liter). Three weeks after treatment,plants with an edited allele showed normal phenotype, while plants withonly wild-type alleles demonstrated strong signs of senescence. Inaddition, embryos isolated from seed derived from plants pollinated withwild-type HI-II pollen were germinated on media with 100 ppm ofchlorsulfuron. Fourteen days after germination, plants with editedalleles showed normal height and a well-developed root system, whileplants with wild-type alleles were short and did not develop roots.

In the above experiment, when ODP2 and WUS2 expression cassettes (on twoseparate plasmids) were not included with the plasmids containing therepair templates, Cas9. ALS-CR4 gRNA, and MoPAT-DsRED, no events wererecovered after particle bombardment of 1000 immature embryos andselection on bialaphos in the Pioneer inbred PHH5G (negative control).By comparison, when plasmids containing PLTP PRO::ODP2::PINII and AXIG1PRO::WUS2::PINII TERM were added to the plasmid mixture for goldparticle preparation and particle bombardment, events containingCAS/CRISPR-mediated gene edits to the ALS gene were recovered. Afterparticle bombardment of approximately 1000 immature embryos from thePioneer inbred PHH5G, over 1000 bialaphos-resistant plantlets wererecovered, and of these, nine were determined to contain edits to thegenomic ALS2 gene conferring resistance to the herbicide chlorsulfuron.

Example 11: High Efficiency Transformation and Rapid to Plant Productionin Sorghum

Sorghum transformation was performed using an optimizedAgrobacterium-mediated protocol (Wu et al., 2014, In Vitro Cellular andDevelopmental Biology 50:8-14) with the methods being summarized below.

A. Sorghum Material and Transformation Process.

TX430, a non-tannin sorghum variety, was used in this study. Greenhousetemperatures averaged 29° C. during the day and 20° C. at night with a12 h d/night photoperiod and supplemental lighting was provided by a 3:1ratio of metal halide (1,000 W) and high-pressure sodium (1,000 W)lamps. The components of the media used in this study are listed inTable 2. The baseline transformation protocol is described in detail as“treatment C” in Zhao et al. (Plant Mol. Biol. (2000) 44:789-798).Briefly, freshly harvested sorghum immature grains were sterilized with50% bleach and 0.1% Tween-20 for 30 min under vacuum and then rinsedwith sterile water three times. The embryos were subjected to thefollowing five sequential steps: (1) Agrobacterium infection: embryoswere incubated in an Agrobacterium suspension (OD=1.0 at 550 nm) withPHI-I medium for 5 min; (2) co-cultivation: embryos were cultured onPHI-T medium following infection for 3 d at 25° C. in the dark; (3)resting: embryos were cultured on PHI-T medium plus 100 mg/lcarbenicillin for 7 d at 28° C. in the dark; (4) selection: embryos werecultured on PHI-U medium for 2 wk, followed by culture on PHI-V mediumfor the remainder of the selection process at 28° C. in the dark, usingsubculture intervals of 2-3 wk; (5) regeneration: callus was cultured onPHI-X medium for 2-3 wk in the dark to stimulate shoot development,followed by culture for 1 wk under conditions of 16 h light (40-120 μEm⁻²s⁻¹) and 8 h dark at 25° C., and a final subculture on PHI-Z mediumfor 2-3 wk under lights (16 h, 40-120 μE m⁻² s⁻¹) to stimulate rootgrowth. Regenerated plantlets were transplanted into soil and grown inthe greenhouse (Zhao et al. 2000). To plants were self-pollinated toproduce T₁ progeny for further analysis.

In another experiment, the embryos were subjected to the following fivesequential steps: (1) Agrobacterium infection with PHP79023: embryoswere incubated in an Agrobacterium suspension (OD=1.0 at 550 nm) withPHI-1 medium for 5 min; (2) co-cultivation: embryos were cultured onPHI-T medium following infection for 7 d at 25° C. in the dark; (3)selection during maturation: embryos were matured on 289M with 0.1 mg/iimazapyr medium for 4 wk, followed by rooting on medium 13113A with 0.1mg/l imazapyr (Medium 13113A contains half-strength MS salts andvitamins, 0.05 g/l myo-inositol, 20 g/i sucrose, and 3 g/l phytagel,pH5.6). Regenerated plantlets were transplanted into soil and grown inthe greenhouse (Zhao et al. 2000). To plants were self-pollinated toproduce T₁ progeny for further analysis.

B. Agrobacterium Strains and Vectors Used with Sorghum.

Agrobacterium tumefaciens strains LBA4404 (Lazo et al. (1991)Biotechnology 9:963-967) was used, containing a first plasmid containingAgrobacterium vir genes (Komari (1990) Plant Cell 9:303-306; Komari etal. (1996) Plant J 10:165-174) and a second plasmid (PHP80334)containing a T-DNA with the following components: RB+ZM-AXIG1PRO::ZM-WUS2::IN2-1 TERM+ZM-PLTP PRO::ZM-ODP2::OS-T28 TERM+GZ-W64ATERM+UBI PRO:UBI1ZM INTRON:ESR::SB-SAG12 TERM+SB-ALS PRO:: HRA::SB-PEPC1TERM+UBI PRO:UBI1ZM INTRON:ZS-GREEN1::PINII TERM:SB-ACTIN TERM-LB. Forcomparison, another Agrobacterium was used that contained a secondplasmid with only a UBI PRO:UBI1 ZM INTRON:PMI::PINII TERM expressioncassette in the T-DNA (with no ZM-WUS2 or ZM-ODP2 expression cassettes)which represents the control treatment in this experiment.

C. Sorghum Transformation Results.

The overall transformation frequency was 20% when the control plasmidwas used, and of the T0 plants sent to the greenhouse and analyzed usingqPCR, 40% were single-copy with no Agrobacterium backbone. For thecontrol treatment, the duration from the start of Agrobacteriuminfection until T0 plants were sent to the greenhouse varied from 10-13weeks.

The overall transformation frequency was 32% when the plasmid containingthe Zm-PLTP PRO::ZM-ODP2+DR5 PRO::ZM-WUS2 (see PHP79023 in Table 1) wasused, and of the T0 plants sent to the greenhouse and analyzed usingqPCR, 42% were single-copy with no Agrobacterium backbone, proceedingdirectly to somatic embryo formation from the originally-infectedzygotic immature embryos, followed by somatic embryo maturation, invitro germination and root formation. Using this combination ofpromoters and developmental genes substantially reduced the time fromAgrobacterium infection to sending T0 plants to the greenhouse byapproximately half relative to the control treatment.

In an additional experiment using the PHP82240 plasmid with a T-DNAcontaining RB-ZM-AXIG1 PRO::ZM-WUS2::IN2-1 TERM+ZM-PLTPPRO::ZM-ODP2::OS-T28 TERM+SB-ALS PRO::ZM-HRA::PINII TERM+a constitutiveDsRED expression cassette-LB, it was demonstrated that direct formationof sorghum somatic embryos was improved by use of PHP82240 with sevendays of co-cultivation on 710I media with 50 mg/L carbenicillin. Thiscombination of plasmid and the seven day co-cultivation resulted indoubling the overall transformation frequency from 17.7% to a finalvalue of 38.9%, while reducing the time from Agrobacterium infection toT0 plants to the greenhouse by approximately half relative to thecontrol treatment (reducing this timeframe to 5-6 weeks total elapsedtime).

Example 12: High Efficiency Transformation and Rapid to Plant Productionin Wheat A. Wheat Transformation Methods.

An aliquot of Agrobacterium strain LBA4404 containing the vector ofinterest was removed from storage at −80° and streaked onto solid LBmedium containing a selective agent (kanamycin or spectinomycin,depending on which plasmids the bacterial strain contains). TheAgrobacterium was cultured on the LB plate at 21° C. in the dark for 2-3days, at which time a single colony was selected from the plate,streaked onto an 810D medium plate containing the selective agent andthen incubated at 28° C. in the dark overnight. The Agrobacteriumculture was transferred from the plate using a sterile spatula andsuspended in ˜5 mL wheat infection medium (WI4) with 400 uMacetosyringone (AS). The optical density (600 nm) of the suspension wasadjusted to about 0.1 to 0.7 using the same medium.

Four to five spikes containing immature seeds (with 1.4-2.3 mm embryos)were collected, and the immature embryos were isolated from the immatureseeds. The wheat grains were surface sterilized for 15 min in 20% (v/v)bleach (5.25% sodium hypochlorite) plus 1 drop of Tween 20, followedwith 2-3 washes in sterile water. The remaining protocol wheat includinginfection with Agrobacterium, co-cultivation, culture during the restingperiod, somatic embryo maturation and rooting, as described in Examples4 and 5 for were followed, including the step of selection on 0.1 mg/limazapyr and maturation for production of transgenic T0 wheat plants.

B. Wheat Transformation Results.

Agrobacterium tumefaciens strain LBA4404 (Lazo et al. 1991,Biotechnology 9:963-967) was used, which contained a first super binaryhelper plasmid (pVIR9, U.S. Provisional Appl. No. 62/252,229, hereinincorporated in entirety by reference) and a second plasmid (PHP79066),with the developmental genes containing a T-DNA with the followingcomponents; RB+ZM-AXIG1 PRO::ZM-WUS2::1N2-1 TERM+ZM-PLTPPRO::ZM-ODP2::OS-T28 TERM+SB-ALS PRO:: HRA::SB-PEPC1 TERM+LTP2PRO::ZS-YELLOW1 N1::PINII TERM+LB. For comparison, another Agrobacteriumwas used that contained a second plasmid (PHP24600) with the followingT-DNA: RB+CAMV35S TERM: PAT: CAMVS PRO+ UBIZM PRO: DS-RED: PINIITERM+LB, which represented the control treatment in this experiment.

When the control plasmid was used to transform wheat immature embryosfrom Pioneer elite Spring wheat variety HC0456D, no regenerablestructures were observed in the first 7-10 days after Agrobacteriuminfection, while immature embryos that were infected with thedevelopmental gene construct PHP79066 produced regenerable structures7-10 days post-infection using maize culture medium described inExamples 4 and 5. The embryos infected with construct PHP79066 producedplant-like structures within 2 weeks (FIG. 5), as compared toproliferating callus observed for the control construct (not shown).Rooted plantlets were recovered after transformation with PHP79066within 6 weeks post infection.

When the plasmid containing the ZM-AXIG1 PRO::ZM-WUS2::1N2-1TERM+ZM-PLTP PRO::ZM-ODP2::OS-T28 TERM+SB-ALS PRO:: HRA::SB-PEPC1TERM+LTP2:ZS-YELLOW::PINII TERM was used for transformation, the overalltransformation frequency was 64% (compared to 6% for the control). Asubset of four plants was analyzed using qPCR, and three were positivefor both WUS and the ZS-YELLOW transgene. Using this combination ofdevelopmental genes, T0 plants regenerated in 8-11 weeks post infection,reducing the time from Agrobacterium infection to sending T0 plants tothe greenhouse by two-three weeks.

Example 13: Observations of Rapid De Novo Embryo Formation in Corn andProduction of to Plants A. Morphology of Developing Somatic Embryos.

After transformation of immature embryos from the Pioneer inbred PH184Cwith the Agrobacterium strain LBA4404 harboring a T-DNA containing theexpression cassettes ZM-AXIG1 PRO::ZM-WUS2::1N2-II TERM+ZM-PLTPPRO::ZM-ODP2::PINII+UBI PRO: UBI1ZM INTRON:ZS-GREEN::PINII TERM, somaticembryos rapidly formed on the surface of the scutellum (i.e. within 4-6days). Directly-formed, single somatic embryos were observed at 2, 4, 7,and 12 days after the beginning of Agrobacterium infection. In InbredPH184C, when the combination of NOS PRO::ZM-WUS2::PINIITERM+ZM-PLTP::ODP2::PINII TERM was used, the single somatic embryos wereobserved as attached to the scutellum of the originally-transformedzygotic embryo by a thin tether of cells that appeared to recapitulatethe suspensor, As embryo development continued, additional morphologicalfeatures were observed that are normally observed in zygotic embryos,including formation of a distinctive coleoptilar ring around the apicalmeristem at the inception of apical meristem development, and formationof the scutellum (which turns white and opaque) surrounding the embryoaxis.

To further illustrate the morphology of the rapidly-forming somaticembryos, zygotic embryos with protruding nascent somatic embryos weresampled at 2, 4, 6, and 7 days after the beginning of Agrobacteriuminfection, and were fixed in 2.5% EM-grade glutaraldehyde in 100 mMphosphate buffer (pH 7.0) at room temperature with rotation at 100 rpmfor 4 hours. After washing with 100 mM phosphate buffer (pH 7.0) threetimes for 15 minutes each, the samples were dehydrated in a step-wisefashion, 1-2 hours in 70% EtOH, 1-2 hours in 80% EtOH, 1-2 hours in 95%EtOH, 1-2 hours in 100% EtOH, and 2 hours in 100% EtOH. The tissue wasthen infiltrated with activated Technovit 7100 glycolmethacrylatefollowing the manufacturer's recommendation, for 2 hours, and again withrefreshed activated Technovit 7100 overnight. The infiltrated tissuesamples were placed in molds, and the Technovit 7100 was polymerized bythe addition of 1 ml Hardener II into 15 mls of activated Technovit7100. The molds were placed under house vacuum in a vacuum desiccatorand allowed to polymerize overnight. Semi-thin 2 μm sections were placedon drops of water on glass slides and dried on a heating plate at 45° C.The sections were stained with periodic acid-Schiff reagent (Sigma)according to instructions and were then counter-stained with 0.5%Naphthol Blue Black in 7% acetic acid solution for 5 minutes and washedagain in deionized water. The sections were then mounted in Permountwith glass coverslips and observed.

FIG. 6 through FIG. 11 show a timecourse for HC69 zygotic immatureembryos at various days after Agrobacterium infection with LBA4404 witha T-DNA which contained the following: RB-AXIG1 PRO::ZM-WUS2::IN2-1TERM+ZM-PLTP PRO::ODP2::PINII TERM-LB. One of the earliest indicationsof localized growth stimulation was the observation of anticlinaldivisions in cells on the surface of the infected scutellum (FIG. 6),which was the first visible disruption in this surface layer thatnormally expands via periclinal divisions that accompany embryo growth.Continued localized cell divisions resulted in small clusters of cellsthat began to protrude from the zygotic embryo surface (FIG. 6), andthis growth rapidly produced increasingly large globular (FIG. 8) andglobular/transition-stage (FIG. 8) embryos, which after only 4 dayspost-infection contained up to 700-800 cells per somatic embryo, andexhibited the typical smooth epidermal layer normally observed inpro-embryos, and with no vascular connections with the underlyingzygotic embryo. Even though the developing somatic embryos were observedin close proximity (FIG. 10) they represented indepedently-derivedstructures. Throughout the growth of the somatic embryos in the first6-7 days post-Agrobacterium-infection, a high frequency of mitoticstructures (i.e. cells clearly undergoing prophase, metaphase, anaphaseor cytokinesis) were observed (FIG. 11) which was indicative of theextremely rapid growth rate of these somatic embryos.

B. Staining to Demonstrate Lipid Accumulation in the Developing SomaticEmbryos.

Directly-formed somatic embryos developing on the scutellur surface ofimmature zygotic embryos after Agrobacterium-mediated transformation(with a T-DNA containing AXIG1 PRO::ZM-WUS2::IN2-1 TERM+ZM-PLTPPRO::ODP2::PINII TERM) were sampled at 2.4 and 7 days afterAgrobacterium infection, washed for 5 minutes in 70% isopropanol, andthen stained for 20 minutes in a 0.5% solution of “oil Red O” in 70%isopropanol, washed for 2 minutes in isopropanol and then washed twicein water for 5 minutes each (all at room temperature). At two days afterAgrobacterium infection, very little lipid staining, as evidenced by redcolor forming in the tissue, was present in the zygotic immatureembryos. At four days after starting the Agrobacterium infection,numerous globular structures (newly-formed somatic embryos) were clearlyvisible growing from the surface of the originally-transformed zygoticembryo, and these new somatic embryos clearly stained red indicating theaccumulation of lipids. At seven days, a mixture of structures wasobserved; some that were clearly somatic embryos that were continuing todevelop and stained red (black arrows in FIG. 12), and some somaticembryos that were beginning to differentiate meristems and leaves whichwere already beginning to lose the red staining associated with lipidaccumulation (FIG. 12). Also seen in FIG. 12, the scutellum of theoriginal Agrobacterium-infected zygotic embryo was almost devoid oflipid under these culture conditions.

Alternatively, lipid was visualized by placing a somatic embryo betweena cover slip and a slide and applying pressure until a monolayer ofcells was extruded. After using the Oil Red O staining method describedabove, numerous oil droplets were observed scattered within the somaticembryo cells when viewed under the light microscope (FIG. 13).

Lipid can be detected using similar staining protocols in sectionedsomatic embryos. Directly-formed, single somatic embryos are sampled at2, 4, 8, and 12 days after the beginning of Agrobacterium infection, andare fixed, dehydrated, infiltrated with plastic and sectioned asdescribed above. For staining for lipids, a 0.5% “Oil Red O” solution in60% triethyl phosphate (aqueous) is prepared and filtered. The tissuesections on the slide are rinse briefly with 60% triethyl phosphate, andthen stained for 10-20 minutes in “Oil Red O”. After staining, thesections are rinsed again with triethyl phosphate for 1-2 seconds andthen washed with distilled, deionized water. The sections are thencounter-stained with 0.5% Celestine Blue in 5% aqueous ferric ammoniumsulfate for 15 minutes and washed again in DI water. The sections arethen mounted using aqueous mounting medium and observed. In thedeveloping somatic embryos, the accumulating lipids in the scutellumappear red, and over the entire section (including the scutellum andembryo axis) the nuclei in each cell will stain blue.

Example 14: Transformation Using WUS2 Alone. ODP2 Alone, Or WUS/ODP2 inCombination

Immature embryos were harvested from three Pioneer inbreds (PH184C, HC69and PHH5G) and the embryos from each inbred were evenly aliquoted fortransformation with Agrobacterium strain LBA4404 containing one thefollowing PHP79066 (SEQ ID NO: 27), PHP80912 (SEQ ID NO: 53), andPHP80913 (SEQ ID NO: 54; each are described in detail in Table 1).

After Agrobacterium-mediated transformation, the three different inbredsresponded differently to the different transgene combinations (Table12). For inbred PH184C, 230 immature embryos were infected for eachtreatment, with 84, 10, and 100% of the originally-infected zygoticembryos producing somatic embryos on the scutellar surface whenexpressing either AXIG1::WUS2 alone, PLTP::ODP2 alone, or thecombination of both WUS2 and ODP2, respectively. After 7 days, all theembryos were moved to maturation and then germination media, with 68, 8and 52 embryos producing T0 plants for the three respective treatments(T0 transformation effieincies of 30%, 3% and 23%, respectively). Forinbred HC69, 80 immature embryos were used for each treatment, withsomatic embryos and T0 plants being efficiently produced in all threetreatments, producing final transgenic T0 plant frequencies (relative tothe number of starting embryos) of 85%, 56% and 68% for the AXIG1::WUS2,PLTP::ODP2, or the combined WUS2+ODP2 treatments, respectively. Finally,for the inbred PHH5G, 168 immature embryos were infected for each of thethree treatments, with final transgenic T0 plant frequencies (relativeto the number of starting embryos) of 27%, 0% or 48% for theAXIG1::WUS2, PLTP::ODP2, or the combined WUS2+ODP2 treatments,respectively. These data demonstrated that although the inbredsexhibited different responses to the WUS alone, ODP2 alone, or thecombination, expression of the individual transcription factors wereeffective in producing transgenic somatic embryos and T0 plants.

TABLE 12 Transformation response in three inbred after delivery of WUS2alone, ODP2 alone, or the combination of WUS2 with ODP2 Embryos Embryosproducing (# producing Somatic a plant; Trans. Embryos Plasmid EmbryosEmbryos one plant Efficiency Inbred (#) No. DevGene (%) (# regen.) perembryo) (T0 level) PH184C 230 PHP80913 Axigi:op:WUS2 84% 230 68 30%PH184C 230 PHP80912 PLTP:ODP2 10% 230 8  3% PH184C 230 PHP79066Axig1:op:WUS2 + 100%  230 52 23% PLTP:ODP2 HC69 80 PHP80913Axigi:op:WUS2 69% 80 68 85% HC69 80 PHP80912 PLTP:ODP2 65% 80 45 56%HC69 80 PHP79066 Axig1:op:WUS2 + 100%  80 54 68% PLTP:ODP2 PHH5G 168PHP80913 Axigi:op:WUS 57% 168 46 27% PHH5G 168 PHP80912 PLTP:ODP2  0%168 0  0% PHH5G 160 PHP79066 Axig1:op:WUS2 + 91% 160 77 48% PLTP:ODP2

Example 15: Improved Transfer of Plantlets Produced from Single SomaticEmbryos to the Greenhouse

Immature embryos (9-11 DAP) of three genotypes (HC69, PHH5G, and PH184C)were infected with PHP79066. Embryos were transferred to co-cultivationmedia (710I) for 1-3 days, resting media 605G (605J media+2 mg/lmeropenem) for 7 days, and then onto maturation media 13329 (289O+0.1mg/l imazapyr). After 3-4 weeks on maturation media, strong singleshoots were transferred to EXcel Plugs (40/80) (InternationalHorticulture Technologies, LLC, 2410 Airline Hwy, Hollister, Calif.,95023) and sprayed with Moisturin (WellPlant, Inc., 940 Spice IslandsDrive, Sparks, NV, 89431). The shoots were cultured under Valoya (ValoyaOy. Lauttasaarentie 54A, 00200 Helsinki, Finland) R-series NS2 LEDlights (220-305 μmol/m²/sec) at 27° C. for 2-3 weeks. Plugs were wateredwith 6N salts and vitamins plus 0.1 mg/L imazapyr every 2-3 days asneeded. After the establishment of roots, the plugs were transferred tothe T0 receiving greenhouse and grown for an additional two weeks. Atthe conclusion of this time frame, survival data were collected for eachof the genotypes and is given below in Table 13.

TABLE 13 Improved survival of T0 plants transferred to the greenhouse.Genotype Shoots* Plantlets** PH184C 54 37 (68.5%) PHH5G 36 17 (47.2%)HC69 36 27 (75.0%) *Total number of shoots transferred to Excel plugs.**Number of plantlets that survived at the conclusion of the experiment,with percentage survival given in parentheses.

Example 16: Results on Use of Sorghum PLTP Promoter to Directly ProduceSomatic Embryos in Corn

The Sorghum bicolor PLTP promoter (SB-PLTP PRO) is provided as SEQ IDNO:2. Immature embryos from Pioneer inbred PH184C or PHH5G weretransformed with the Agrobacterium strain LBA4404 harboring the plasmidRV012608 containing a T-DNA (SEQ ID NO:95), RB+ZM-AXIG1PRO::ZM-WUS2::IN2-1 TERM+SB-PLTP1 PRO::ZM-ODP2:: OS-T28 TERM+GZ-W64ATERM+UBI PRO:UBI1ZM INTRON:ESR::SB-SAG12 TERM+SB-ALS PRO:: HRA::SB-PEPC1TERM+UBI PRO::ZS-GREEN1::PINII TERM:SB-ACTIN TERM-LB. Many discreetsomatic embryos formed on the surface of the inbred zygotic embryosafter 4-6 days in culture. These early somatic embryos were clearlydistinct from one another (i.e. with intervening non-transgenic tissuebetween the forming somatic embryos) and were confirmed to be transgenicbased on expression of the green fluorescence protein. When theseembryos were transferred to maturation medium with 0.1 mg/I imazapyr theembryos continued to develop, and when transferred to germinationmedium, both shoot and root elongation occurred.

Example 17: Results Using Maize ODP2 Homologs, WUS2 Homologs, and PLTPPromoters from Homologous Gene-Sources to Produce Somatic Embryos inCorn

A list of WUS/WOX paralogs (family member genes and encoded proteins)and ODP2/BBM family members and their corresponding SEQ ID Numbers areshown in Table 14 below.

TABLE 14 WUS/WOX and ODP2/BBM Sequences SEQ ID NO. Type* NameDescription 3 DNA ZM-WUS1 Z. mays WUS1 coding sequence 4 PRT* ZM-WUS1 Z.mays WUS1 protein sequence 5 DNA ZM-WUS2 Z. mays WUS2 coding sequence 6PRT ZM-WUS2 Z. mays WUS2 protein sequence 7 DNA ZM-WUS3 Z. mays WUS3coding sequence 8 PRT ZM-WUS3 Z. mays WUS3 protein sequence 9 DNAZM-WOX2A Z. mays WOX2A coding sequence 10 PRT ZM-WOX2A Z. mays WOX2Aprotein sequence 11 DNA ZM-WOX4 Z. mays WOX4 coding sequence 12 PRTZM-WOX4 Z. mays WOX4 protein sequence 13 DNA ZM-WOX5A Z. mays WOX5Acoding sequence 14 PRT ZM-WOX5A Z. mays WOX5A protein sequence 15 DNAZM-WOX9 Z. mays WOX9 coding sequence 16 PRT ZM-WOX9 Z. mays WOX9 proteinsequence 17 DNA ZM-ODP2 Z. mays ODP2 coding sequence 18 PRT ZM-ODP2 Z.mays ODP2 protein sequence 19 DNA ZM-BBM2 Z. mays BBM2 coding sequence20 PRT ZM-BBM2 Z. mays BBM2 protein sequence 21 DNA ZM-ODP2 Z. mays ODP2coding sequence (synthetic) *“DNA” indicates a polynucleotide or nucleicacid sequence; “PRT” indicates a polypeptide or protein sequence.

For these studies described in sections A, B and C below, a single T-DNAconfiguration was utilized (SEQ ID NO: 104), starting with the followingpositive control: RB+ZM-AXIG1 PRO::ZM-WUS2::IN2-1 TERM+ZM-PLTPPRO::ZM-ODP2:: OS-T28 TERM+GZ-W64A TERM+UBI PRO:UBI1ZMINTRON:ESR::SB-SAG12 TERM+SB-ALS PRO:: HRA::SB-PEPC1 TERM+UBIPRO::ZS-GREEN1::PINII TERM:SB-ACTIN TERM-LB. Within the context of thisT-DNA, all the components of the T-DNA remained the same except forthree variables described below. In the first variable, ZM-WUS2 (in thecontrol plasmid) was replaced by either ZM-WOX2A, ZM-WOX4, ZM-WOX5A, orthe sorghum (SB) WUS1. In the second variable. ZM-PLTP PRO (from thecontrol treatment) was replaced by promoters from two maize paralogs(ZM-PLTP1 and ZM-PLTP2) or from three Poaceae orthologs (Sorghum bicolorSB-PLTP1, Setaria italica SI-PLTP1 or Oryza sativa OS-PLTP1). When thecontrol T-DNA (all maize components as shown above) was introduced intothe scutellum of Pioneer inbreds PH1V5T, PH1V69 and PHH5G, approximatelyhalf of the scutellar surface area was covered by newly developedsomatic embryos after 7 days. This response was scored as a “2”. At theupper end of the response spectrum the scutellum was completely coveredby a lawn of individual, developing somatic embryos 4-7 dayspost-infection. This response was given a relative score of “4” and allother treatments were ranked from “0” (no response) to “4” (the mostprolific production of somatic embryos). When no WUS2 or ODP2 expressioncassettes were introduced, for example in PHP24600, SEQ ID NO:69),PH1V5T produced a low level of somatic embryos (score of 1), while bothPH1V69 and PHH5G produced no response (score of 0).

A. Substitution of ZM-WOX family members or a sorghum WUS1 for ZM-WUS2produced varying degrees of rapidly formed somatic embryos.

In this experiment, a positive control plasmid, containing the ZM-AXIG1PRO::ZM-WUS2+ZM-PLTP PRO::ZM-ODP2 in the T-DNA (SEQ ID NO; 104) producedan intermediate (score of 2) response in inbreds PH1V5T and PHH5G, whileproducing a lower score of “I” in inbred PH1V69. By comparison, whenthree maize WOX family members were substituted for ZM-WUS2, a range ofsomatic embryogenesis responses was observed (Table 15), ranging from ahigher somatic embryo response from ZM-WOX2A, a low-to-no response forZM-WOX4 and a low response for WOX5 (T-DNA sequences provided in SEQ IDNO:100, SEQ ID NO:101 and SEQ ID NO:102, respectively). Even though WOX5and WOX4 resulted in fewer somatic embryos on the surface of thetransformed immature embryo, there was still a positive response for allthree inbreds with WOX5 and a subset of inbreds for WOX4. For treatmentsthat produced a low level response, the inbred PH1V5T was difficult tointerpret because it exhibited low levels of growth in the absence ofWUS2 and ODP2 expression cassettes. However, for such treatments theother two inbreds became much more informative because they exhibited nobackground growth and a low level response (1) was unequivocal.

TABLE 15 Inbred transformation response to different WUS homologsZM-AXIG1 ZM-PLTP with constant for Response in Inbreds WUS/WOX ZM-ODP2PH1V5T PH1V69 PHH5G ZM-WUS2 PLTP::ODP2 2 1 2 ZM-WOX2a PLTP::ODP2 3 3 2ZM-WOX4 PLTP::ODP2 1 1 0 ZM-WOX5 PLTP::ODP2 2 1 1 SB-WUS1 PLTP::ODP2 4 44

ZM-WUS1 and ZM-WUS3 were also compared to ZM-WUS2 for stimulation ofrapid growth responses after transformation. In this experiment, aplasmid containing UBI::GFP::PINII TERM was co-bombarded using astandard particle bomardment protocol for maize (see Svitachev et al.,2015, Plant Physiology 169:931-945) in equimolar ratios along with aplasmid containing either UBI PRO::WUS1::PINII TERM, UBIPRO::WUS2::PINII TERM or UBI PRO::WUS3::PINII TERM. Embryos wereobserved under a Leica Mzfl III epifluorescence microscope with a GFPfilter. After 7 days, the surface of the scutellum that was bomarded wascovered with rapidly growing multicellular structures with GFPfluorescence at the center. For all three WUS3 paralogs, the growth ratewas fast, the response was extensive across the surface of the bombardedscutellum, and no differences could be discerned between the three WUSparalogs. Based on these observation, one would expect that ZM-WUS1 andZM-WUS3 would produce a similar degree of rapid somatic embryogenesis asZM-WUS2 when placed behind the AXIG1 promoter and combined in a T-DNAwith ZM-PLTP PRO::ZM-ODP2 for transformation into maize immatureembryos.

A final treatment in this experiment was to substitute the sorghum (SB)WUS1 (T-DNA sequence provided as SEQ ID NO:103) for the maize WUS2. Thistreatment produced the most rapid and prolific somatic embryo responseof any treatment, with approximately 80% of the infected embryos beingentirely covered with somatic embryos.

B. Substitution of PLTP promoters from maize paralogs or three differentPoaceae orthologs resulted in varying degrees of rapid somaticembryogenesis.

Using various “homologous” promoters produced a range of rapid somaticembryogenesis in three different Pioneer inbreds (Table 16) relative tothe control treatment (ZM-PLTP PRO), in which the control producedscores between 1 and 2.

TABLE 16 Inbred transformation response to different PLTP promoterhomologs ZM-AXIG1 Promoter constant for for Response in Inbred ZM-WUS2ZM-ODP2 PH1V5T PH1V69 PHH5G Zm-Axig1 ZM-PLTP 2 1 2 Zm-Axig1 ZM-PLTP1 3 44 Zm-Axig1 ZM-PLTP2 3 3 3 Zm-Axig1 SB-PLTP1 2 2 1 Zm-Axig1 SI-PLTP1 1 12 Zm-Axig1 OS-PLTP1 1 2 2

In this experiment, the ZM-PLTP1 promoter produced the highest somaticembryogenesis scores at seven days post-infection, which ranged from 3(roughly 75% covered with somatic embryos in PH1V5T) to 4 (totallycovered as in PH1V69 and PHH5G). ZM-PLTP2 also produced results betterthan the control, with a uniform score of 3 across all three inbreds.For PLTP1 promoters from other members of the Poaceae, the sorghum andrice promoters produced an intermediate level response (2) in twoinbreds and a low response (1) in one inbred, while the Setaria promoterresulted in a low level response in two inbreds and an intermediatelevel response in one inbred. All PLTP promoters tested resulted inpositive stimulation of somatic embryogenesis after seven days.

C. ZM PLTP PRO::ZM-LEC1 in combination with either AXIG1::WUS2 orPLTP::ODP2 resulted in rapid production of somatic embryos.

In previous experiments it was demonstrated that UBI PRO::ZM-LEC1stimulated transformation frequencies in the transformable maize hybridHi-II (see Lowe et al., 2007, U.S. Pat. No. 7,268,271).

In this experiment ZM-PLTP::ZM-LEC1 was substituted for eitherAXIG1::WUS2 or PLTP::ODP2 in a control vector to assess the impact onrapid somatic embryogenesis in three inbreds. Conditions forAgrobacterium infection, tissue culture and scoring for embryogenesis atseven days were the same as those described in earlier examples. Whileboth of the new combinations produced higher scores than the control, ascan be seen in Table 17, AXIG1::WUS2+PLTP::LEC1 resulted in thestrongest somatic embryo response across all three inbreds. Thus,PLTP::LEC1 in combination with either WUS2 or ODP2 expression cassetteswas effective at stimulating rapid formation of somatic embryos.

TABLE 17 Inbred transformation response to different combinations withLEC1 Combinations Response for Inbreds with LEC1 PH1V5T PH1V69 PHH5GPLTP::LEC1 + 1 3 2 PLTP::ODP2 AXIG1::WUS2 + 3 3 2 PLTP::LEC1AXIG1::WUS2 + 2 1 2 PLTP::ODP2D. Use of rice WUS and ODP2 in combination or Setaria WUS and ODP2 incombination resulted in somatic embryo formation after transformation ofmaize immature embryos.

Starting with a maize construct containing NOS PRO::ZM-WUS2::IN2+UBIPRO::ZM-ODP2::PINII+UBI PRO::ZS-GREEN::PINII, orthologs for WUS2 andODP2 identified in Oryza sativa and Setaria italica were synthesized andsubstituted for the maize genes in the above construct. Immature embryosfrom inbred PHH5G and PH184C were transformed using Agrobacterium strainLBA4404 containing either PHP80911 (maize WUS2 and ODP2), PHP79530 (ricegenes) or PHP79531 (foxtail millet genes). After 14 days on culturemedium, the immature embryos were examined under the dissectingmicroscope and the epifluorescence streo-microscope and scored using thesomatic embryogenesis scale (0-4), previously described. For inbredPH184C, the somatic embryo scores 14 days after Agrobacterium infectionwere 2, 2 and 1 (for the maize, millet and rice gene-pairs,respectively). For inbred PHH5G, the somatic embryo scores 14 days afterAgrobacterium infection were 3, 3 and 2 (for the maize, millet and ricegene-pairs, respectively). These immature embryos were exposed toexpression of WUS2 and ODP2 for twice the length of time as was thematerial in sections 5A, 5B and 5C above, which resulted in a highersomatic embryogenesis score than if the data had been collected at 7days. The combinations of the cognate WUS and ODP2 genes from eachspecies stimulated somatic embryo formation. In the absence of eitherWUS2 or ODP2, no somatic embryo formation was observed in PHH5G.

Within the Poaceae, maize and rice are at opposite ends of the phylogenywith millet in the middle. Based on the results with the above describeddivergent sequences for WUS, ODP2 and PLTP, this work demonstrated thatcombinations from members across the grasses can effectively be used torapidly stimulate somatic embryogenesis after transformation.

Example 18. Use of the Soybean LTP3 Promoter to Control Expression ofWUS for Improving Soy Transformation

In order to identify new promoters that might improve transformationmethods using the Arabidopsis WUS gene, the use of this gene wasreviewed. High levels of expression for Arabidopsis WUS (for example,using the soybean EF1A PRO), expressed immediately afterAgrobacterium-mediated transformation and throughout callus growthincreased the rates of event formation. However, continuing to expressthis transcription factor at this level hindered event regeneration.Possible solutions would be to excise this gene before regeneration ofplantlets and restrict the ectopic expression of Arabidopsis WUS indifferentiating and maturing somatic embryos. Based on this, newpromoters were sought that expressed in cultured cells, embryos anddeveloping immature seeds, with none or much lower expression in otherplant tissues. The soybean LTP3 promoter met these criteria, apreviously unidentified soybean phospholipid transferase gene. As shownin FIG. 14 and FIG. 15, when compared to the constitutive expression ofthe EF1A PRO (FIG. 14), expression of LTP3 (FIG. 15) was i) strong indeveloping immature seeds and ii) weak or off in other samples and partsof a plant, while expression of EF1A was observed in all tissues.

The Agrobacterium strain AGL1, containing a T-DNA with the expressioncassettes GM-LTP3 PRO::AT-WUS::UBI14 TERM+GM-UBQ PRO::TAGRFP::UBQ3 TERM,was used to transform the Pioneer soybean variety PHY21. Four days afterthe Agrobacterium infection was started, the tissue was washed withsterile culture medium to remove excess bacteria. Nine days later thetissue was moved to somatic embryo maturation medium, and 22 days laterthe transgenic somatic embryos were ready for dry-down. At this point,well-formed, mature somatic embryos were fluorescing red under anepifluorescence stereo-microscope with an RFP filter set. The somaticembryos that developed were functional and germinated to produce healthyplants in the greenhouse. This rapid method of producing somatic embryosand germinating to form plants reduced the typical timeframe fromAgrobacterium infection to moving transgenic T0 plants into thegreenhouse from four months (for conventional soybean transformation) totwo months.

As shown in the box plot diagram in FIG. 16 which displays thedistribution of somatic embryogenesis responses of immature cotyledonexplants 2 weeks after Agrobacterium infection, the use of the LTP3promoter to drive expression of At-WUS resulted in a substantialimprovement in somatic embryogenesis (as compared to other promoterstested, see FIG. 16) or to the negative control with no WUS expressioncassette (see FIG. 16).

The increase in somatic embryo response across the population ofinfected immature cotyledons was also accompanied by rapid somaticembryo development, which was observed under both light microscopy toassess morphology (FIG. 17A) and epifluorescence to observe redfluorescence (FIG. 17B). It shows mature transgenic soybean somaticembryos that were ready for desiccation and thereafter germination only5 weeks after Agrobacterium infection. When immature cotyledons weretransformed without LTP3::At-WUS (control treatment) mature somaticembryos were not only produced at a greatly reduced frequency (see FIG.16) but the duration from Agrobacterium infection to a comparable stageof somatic embryo maturity required nine weeks of culture.

Example 19. Excision-Activated Selectable Marker HRA to Increase theRecovery of to Plants in which the WUS2. ODP2 and Cre ExpressionCassettes were Excised and the Remaining Trait Gene was Single Copy

In order to construct a vector with excision-activated HRA expression,the HRA gene was interrupted by the ST-LS1 Intron containing a singleloxP target site in the center of the intron, and was tested todemonstrate that the loxP-containing intron functioned properly. Twohalves of the HRA expression cassette (split at the loxP site) weremoved to opposite ends of the T-DNA with each half having it's owninternal loxP site, which were closer to the center of the T-DNA thanthe HRA halves. Within the two loxP sites were the AXIG1 PRO::WUS2::IN2TERM, the PLTP PRO::ODP2::OS-T28 TERM, ZM-GLB1 PRO::CRE::pinII and theSB-UBI PRO::ZS-GREEN::OS-UBI TERM expression cassettes (see PHP81814 inTable 1, and SEQ ID NO:80).

This construct produced a high frequency of rapid somatic embryoformation with excision of the combined WUS/ODP2/CRE/ZS-GREEN cassettesto activate HRA expression, and recovery of single-copy, T0 plants. Inone set of experiments a combined total of 2332 immature embryos (frominbred PHH5G) were used for transformation by Agrobacterium strainLBA4404 THY-carrying PHP81814. Of the total embryos infected, a total of604 T0 plants were recovered, and of these 604 T0 plants a total of 215no longer contained the WUS2, ODP2, CRE and SZ-GREEN expressioncassettes (totally excised). An overall recovery of quality events of9.2% (number of perfect events relative to the number of startingembryos) resulted.

In a separate set of three experiments with inbred HC69, 741 immatureembryos were transformed with Agrobacterium containing PHP81814, andproduced 315 T0 plants in the greenhouse, of which 30 were single-copyfor HRA with all the other genes excised for a frequency of qualityevents of 4%.

Example 20. Use of Promoters with Embryo-Specific Expression Patterns toDrive Expression of WUS2 and/or ODP2 to Improve Maize Transformation

For these experiments, a single T-DNA configuration is used, startingwith the following configuration used as a positive control: RB+ZM-AXIG1PRO::ZM-WUS2::IN2-1 TERM+ZM-PLTP PRO::ZM-ODP2::OS-T28 TERM+GZ-W64ATERM+UBI PRO:UBI1ZM INTRON:ESR::SB-SAG12 TERM+SB-ALS PRO:: HRA::SB-PEPC1TERM+UBI PRO::ZS-GREEN1::PINII TERM:SB-ACTIN TERM-LB. The positivecontrol is compared to plasmids with embryo specific promoters drivingexpression of WUS2 and ODP2. The plasmid PHP24600 is used as thenegative control (no expression of WUS2 and ODP2 transgenes) to providea lower baseline for comparison.

When the ZM-SRD PRO, the ZM-LGL PRO, the ZM-LEA14-A PRO or theZM-LEA-D-34 promoters (SEQ ID NO:38, SEQ ID NO:81, SEQ ID NO:82 and SEQID NO:83, respectively) are used to replace the ZM-AXIG1 PRO drivingexpression of WUS2 in this construct, increased transformationfrequencies and stimulation of rapid somatic embryogenesis is expectedto be similar to that observed with the positive control vector, bothbeing substantially greater than the negative control treatment(PHP24600). Likewise, when the ZM-SRD PRO, the ZM-LGL PRO, theZM-LEA14-A PRO or the ZM-LEA-D-34 promoters is used to replace theZM-PLTP PRO driving expression of ODP2 in this construct, increasedtransformation frequencies and stimulation of rapid somaticembryogenesis is again expected to be similar to that observed with thepositive control vector, both being substantially greater than thenegative control treatment (PHP24600).

Example 21. Sequence Identification

Various sequences are referenced in the disclosure. Sequence identifiersare found below in Table 18.

TABLE 18 SEQ ID NO. Type* Name Description 1 DNA ZM-PLTP Z. mays PLTPpromoter sequence 2 DNA SB-PLTP1 Sorghum biocolor PLTP1 promotersequence 3 DNA ZM-WUS1 Z. mays WUS1 coding sequence 4 PRT ZM-WUS1 Z.mays WUS1 protein sequence 5 DNA ZM-WUS2 Z. mays WUS2 coding sequence 6PRT ZM-WUS2 Z. mays WUS2 protein sequence 7 DNA ZM-WUS3 Z. mays WUS3coding sequence 8 PRT ZM-WUS3 Z. mays WUS3 protein sequence 9 DNAZM-WOX2A Z. mays WOX2A coding sequence 10 PRT ZM-WOX2A Z. mays WOX2Aprotein sequence 11 DNA ZM-WOX4 Z. mays WOX4 coding sequence 12 PRTZM-WOX4 Z. mays WOX4 protein sequence 13 DNA ZM-WOX5A Z. mays WOX5Acoding sequence 14 PRT ZM-WOX5A Z. mays WOX5A protein sequence 15 DNAZM-WOX9 Z. mays WOX9 coding sequence 16 PRT ZM-WOX9 Z. mays WOX9 proteinsequence 17 DNA ZM-ODP2 Z. mays ODP2 coding sequence 18 PRT ZM-ODP2 Z.mays ODP2 protein sequence 19 DNA ZM-BBM2 Z. mays BBM2 coding sequence20 PRT ZM-BBM2 Z. mays BBM2 protein sequence 21 DNA ZM-ODP2 Z. mays ODP2coding sequence (synthetic) 22 DNA PHP77833 T-DNA sequence (RB to LB) 23DNA PHP78157 T-DNA sequence (RB to LB) 24 DNA PHP78156 T-DNA sequence(RB to LB) 25 DNA PHP79023 T-DNA sequence (RB to LB) 26 DNA PHP79024T-DNA sequence (RB to LB) 27 DNA PHP79066 T-DNA sequence (RB to LB) 28DNA ZM-FBP1 Z. mays promoter for Fructose-1,6-bisphosphatase 29 DNAZM-RFP Z. mays promoter for NAD(P)-binding Rossmann-Fold Protein 30 DNAZM-APMP Z. mays promoter for adipocyte plasma membrane-associatedprotein-like protein 31 DNA ZM-RfeSP Z. mays promoter for Rieske[2Fe—2S] iron-sulphur domain protein 32 DNA ZM-CRR6 Z. mays promoter forChlororespiratory reduction 6 gene 33 DNA ZM-GLYK Z. mays promoter forD-glycerate 3-kinase, chloroplastic-like protein gene 34 DNA ZM-CAB7 Z.mays promoter for Chlorophyll a-b binding protein 7, chloroplastic-likeprotein 35 DNA ZM-UBR Z. mays promoter for Ultraviolet-B-repressibleprotein gene 36 DNA ZM-HBP Z. mays promoter for Soul heme-binding familyprotein 37 DNA ZM-PSAN Z. mays promoter for Photosystem I reactioncenter subunit psi-N 38 DNA ZM-SDR Z. mays promoter for Short-chaindehydrogenase/reductase 39 DNA AXIG1 AXIG1 promoter sequence 40 DNA DR5DR5 promoter sequence 41 DNA PHP80334 T-DNA sequence (RB to LB) 42 DNAPHP80338 T-DNA sequence (RB to LB) 43 DNA PHP38332 T-DNA sequence (RB toLB) 44 DNA PHP80921 T-DNA sequence (RB to LB) 45 DNA OS-BBM1 Oryzasativa BBM1 coding sequence 46 PRT OS-BBM1 Oryza sativa BBM1 proteinsequence 47 DNA OS-BBM2 Oryza sativa BBM2 coding sequence 48 PRT OS-BBM2Oryza sativa BBM2 protein sequence 49 DNA OS-BBM3 Oryza sativa BBM3coding sequence 50 PRT OS-BBM3 Oryza sativa BBM3 protein sequence 51 DNASB-BBM2 Sorghum bicolor BBM2 coding sequence 52 PRT SB-BBM2 Sorghumbicolor BBM2 protein sequence 53 DNA PHP80912 T-DNA sequence (RB to LB)54 DNA PHP80913 T-DNA sequence (RB to LB) 55 DNA ZM-PLTP1 Z. mays PLTP1promoter sequence 56 DNA ZM-PLTP2 Z. mays PLTP2 promoter sequence 57 DNASB-PLTP2 Sorghum bicolor PLTP2 promoter sequence 58 DNA SB-PLTP3 Sorghumbicolor PLTP3 promoter sequence 59 DNA SI-PLTP1 Setaria italica PLTPpromoter sequence 60 DNA OS-PLTP1 Oryza sativa PLTP promoter sequence 61DNA OS-PLTP2 Oryza sativa PLTP2 promoter sequence 62 DNA SB-ODP2 Sorghumbicolor ODP2 coding sequence 63 PRT SB-ODP2 Sorghum bicolor ODP2 proteinsequence 64 DNA SI-ODP2 Setaria italica ODP2 coding sequence 65 PRTSI-ODP2 Setaria italica ODP2 protein sequence 66 DNA BD-ODP2Brachypodium distachyum ODP2 coding sequence 67 PRT BD-ODP2 Brachypodiumdistachyum ODP2 protein sequence 68 DNA SB-ODP2 Sorghum bicolor ODP2genomic sequence 69 DNA PHP24600 Synthetic construct comprising theT-DNA (LB to RB) 70 DNA PHP79530 Synthetic construct comprising theT-DNA (LB to RB) 71 DNA PHP79531 Synthetic construct comprising theT-DNA (LB to RB) 72 DNA PHP80911 Synthetic construct comprising theT-DNA (LB to RB) 73 DNA PHP80334 Synthetic construct comprising theT-DNA (LB to RB) 74 DNA PHP80558 Synthetic construct comprising theT-DNA (LB to RB) 75 DNA PHP80559 Synthetic construct comprising theT-DNA (LB to RB) 76 DNA PHP80561 Synthetic construct comprising theT-DNA (LB to RB) 77 DNA PHP80770 Synthetic construct comprising theT-DNA (LB to RB) 78 DNA PHP81430 Synthetic construct comprising theT-DNA (LB to RB) 79 DNA PHP81431 Synthetic construct comprising theT-DNA (LB to RB) 80 DNA PHP81814 Synthetic construct comprising theT-DNA (LB to RB) 81 DNA ZM-LGL Z. mays promoter for thelactoylglutathione lyase gene (ZM-LGL PRO PRO) 82 DNA ZM-LEA14-A Z. mayspromoter for gene encoding late embryogenic abundant PRO proteinLea-14-A (ZM-LEA14-A PRO) 83 DNA ZM-LEA34-D Z. mays promoter for geneencoding late embryogenic abundant PRO protein Lea34-D (ZM-LEA34-D PRO)84 DNA PHP80560 Synthetic construct comprising the T-DNA (LB to RB) 85DNA PHP82240 Synthetic construct comprising the T-DNA (LB to RB) 86 DNAZM-SDR Z. mays promoter for the short-chain dehydrogenase/reductase PRO(FL) [ZM-SDR PRO (FL)] 87 DNA OS-SDR O. sativa promoter for theshort-chain dehydrogenase/reductase PRO (OS-SDR PRO) 88 DNA SB-SDR S.Bicolor promoter for the short-chain dehydrogenase/reductase PRO (SB-SDRPRO) 89 DNA RV003866 Synthetic construct comprising the T-DNA (LB to RB)90 DNA RV004886 Synthetic construct comprising the T-DNA (LB to RB) 91DNA RV012587 Synthetic construct comprising the T-DNA (LB to RB) 92 DNARV012588 Synthetic construct comprising the T-DNA (LB to RB) 93 DNARV012589 Synthetic construct comprising the T-DNA (LB to RB) 94 DNARV012590 Synthetic construct comprising the T-DNA (LB to RB) 95 DNARV012591 Synthetic construct comprising the T-DNA (LB to RB) 96 DNARV012592 Synthetic construct comprising the T-DNA (LB to RB) 97 DNARV012593 Synthetic construct comprising the T-DNA (LB to RB) 98 DNARV012594 Synthetic construct comprising the T-DNA (LB to RB) 99 DNARV012595 Synthetic construct comprising the T-DNA (LB to RB) 100 DNARV012603 Synthetic construct comprising the T-DNA (LB to RB) 101 DNARV012604 Synthetic construct comprising the T-DNA (LB to RB) 102 DNARV012605 Synthetic construct comprising the T-DNA (LB to RB) 103 DNARV012606 Synthetic construct comprising the T-DNA (LB to RB) 104 DNARV012608 Synthetic construct comprising the T-DNA (LB to RB) 105 DNAPHP80730 Synthetic construct comprising the T-DNA (LB to RB) 106 DNALTP3 G. max promoter for the Lipid Transfer Protein gene (LTP3) *“DNA”indicates a polynucleotide or nucleic acid sequence; “PRT” indicates apolypeptide or protein sequence.

As used herein the singular forms “a”, “an”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, tierexample, reference to “a cell” includes a plurality of such cells andreference to “the protein” includes reference to one or more proteinsand equivalents thereof known to those skilled in the art, and so forth.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisdisclosure belongs unless clearly indicated otherwise.

All patents, publications and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this disclosure pertains. All patents, publications and patentapplications are herein incorporated by reference in the entirety to thesame extent as if each individual patent, publication or patentapplication was specifically and individually indicated to beincorporated by reference in its entirety.

Although the foregoing disclosure has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, certain changes and modifications may be practiced withinthe scope of the appended claims.

1-30. (canceled)
 31. A method for producing a transgenic Poaceae plant,comprising: (a) transforming a cell of a Poaceae explant with anexpression construct comprising a heterologous gene of interest; and (i)a nucleotide sequence encoding a WUS/WOX homeobox polypeptide; and (ii)a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an OvuleDevelopment Protein 2 (ODP2) polypeptide; and (b) allowing expression ofthe expression construct of (a) in each transformed cell for about 0 toabout 7 days or for about 0 to about 14 days after initiation oftransforming the cell, wherein a somatic embryo is formed within about 0to about 7 days or within about 0 to about 14 days after initiation oftransforming the cell; and (c) germinating the somatic embryo to formthe transgenic Poaceae plant.
 32. The method of claim 31, whereingerminating comprises transferring the somatic embryo to a maturationmedium and forming the transgenic Poaceae plant.
 33. The method of claim31, wherein the expression construct further comprises a nucleotidesequence encoding a site-specific recombinase selected from FLP, Cre,SSV1, lambda Int, phi C31 Int, HK022, R, Gin, Tn1721, CinH, ParA,Tn5053, Bxb1, TP907-1, or U153.
 34. The method of claim 33, wherein thenucleotide sequence encoding a site-specific recombinase is operablylinked to a constitutive promoter, an inducible promoter, or adevelopmentally regulated promoter.
 35. The method of claim 31, wherein(c) is performed in the presence of exogenous cytokinin.
 36. The methodof claim 31, wherein the nucleotide sequence encoding the WUS/WOXhomeobox polypeptide and the nucleotide sequence encoding the BBMpolypeptide or the ODP2 polypeptide is operably linked to a promoterselected from an inducible promoter, a developmentally regulatedpromoter, or a constitutive promoter.
 37. The method of claim 34,wherein the constitutive promoter is selected from UBI, LLDAV, EVCV,DMMV, BSV(AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO(ALT1), USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO (1.2 KB), IN2-2, NOS, the-135 version of 35S, or ZM-ADF PRO (ALT2); the inducible promoter isselected from AXIG1, DR5, XVE, GLB1, OLE, LTP2, HSP17.7, HSP26, HSP18A,or promoters activated by tetracycline, ethamethsulfuron orchlorsulfuron; and the developmentally regulated promoter is selectedfrom PLTP, PLTP1, PLTP2, PLTP3, SDR, LGL, LEA-14A, or LEA-D34.
 38. Themethod of claim 36, wherein the constitutive promoter is selected fromUBI, LLDAV, EVCV, DMMV, BSV(AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO,SB-UBI PRO (ALT1), USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO (1.2 KB), IN2-2,NOS, the -135 version of 35S, or ZM-ADF PRO (ALT2); the induciblepromoter is selected from AXIG1, DR5, XVE, GLB1, OLE, LTP2, HSP17.7,HSP26, HSP18A, or promoters activated by tetracycline, ethamethsulfuronor chlorsulfuron; and the developmentally regulated promoter is selectedfrom PLTP, PLTP1, PLTP2, PLTP3, SDR, LGL, LEA-14A, or LEA-D34.
 39. Amethod for producing a transgenic Poaceae plant, comprising: (a)transforming a cell of a Poaceae explant with an expression constructcomprising a heterologous gene of interest; and (i) a nucleotidesequence encoding a WUS/WOX homeobox polypeptide; and (ii) a nucleotidesequence encoding a Babyboom (BBM) polypeptide or an Ovule DevelopmentProtein 2 (ODP2) polypeptide; and (b) allowing expression of theexpression construct of (a) in each transformed cell for about 0 toabout 7 days or for about 0 to about 14 days after initiation oftransforming the cell, wherein a somatic embryo is formed within about 0to about 7 days or within about 0 to about 14 days after initiation oftransforming the cell; and (c) germinating the somatic embryo to formthe transgenic Poaceae plant; wherein the WUS/WOX homeobox polypeptidecomprises the amino acid sequence of any of SEQ ID NO: 4, 6, 8, 10, 12,14, or 16; or wherein the WUS/WOX homeobox polypeptide is encoded by thenucleotide sequence of any of SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15;wherein the polypeptide comprising the BBM polypeptide or the ODP2polypeptide comprises the amino acid sequence of any of SEQ ID NO: 18,20, 63, 65, or 67; or wherein the polypeptide comprising the BBMpolypeptide or the ODP2 polypeptide is encoded by the nucleotidesequence of any of SEQ ID NO: 17, 19, 21, 62, 64, 66, or
 68. 40. Themethod of claim 39, wherein germinating comprises transferring thesomatic embryo to a maturation medium and forming the transgenic Poaceaeplant.
 41. The method of claim 39, wherein the expression constructfurther comprises a nucleotide sequence encoding a site-specificrecombinase selected from FLP, Cre, SSV1, lambda Int, phi C31 Int,HK022, R, Gin, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, or U153. 42.The method of claim 41, wherein the nucleotide sequence encoding asite-specific recombinase is operably linked to a constitutive promoter,an inducible promoter, or a developmentally regulated promoter.
 43. Themethod of claim 39, wherein (c) is performed in the presence ofexogenous cytokinin.
 44. The method of claim 39, wherein the nucleotidesequence encoding the WUS/WOX homeobox polypeptide and the nucleotidesequence encoding a polypeptide comprising the BBM polypeptide or theODP2 polypeptide is operably linked to an inducible promoter, adevelopmentally regulated promoter, or a constitutive promoter.
 45. Themethod of claim 42, wherein the constitutive promoter is selected fromUBI, LLDAV, EVCV, DMMV, BSV (AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3PRO, SB-UBI PRO (ALT1), USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO (1.2 KB),IN2-2, NOS, the -135 version of 35S, or ZM-ADF PRO (ALT2); the induciblepromoter is selected from AXIG1, DR5, XVE, GLB1, OLE, LTP2, HSP17.7,HSP26, HSP18A, or promoters activated by tetracycline, ethamethsulfuronor chlorsulfuron; and the developmentally regulated promoter is selectedfrom PLTP, PLTP1, PLTP2, PLTP3, LGL, LEA-14A, or LEA-D34.
 46. The methodof claim 44, wherein the constitutive promoter is selected from UBI,LLDAV, EVCV, DMMV, BSV(AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO,SB-UBI PRO (ALT1), USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO (1.2 KB), IN2-2,NOS, the -135 version of 35S, or ZM-ADF PRO (ALT2); the induciblepromoter is selected from AXIG1, DR5, XVE, GLB1, OLE, LTP2, HSP17.7,HSP26, HSP18A, or promoters activated by tetracycline, ethamethsulfuronor chlorsulfuron; and the developmentally regulated promoter is selectedfrom PLTP, PLTP1, PLTP2, PLTP3, SDR, LGL, LEA-14A, or LEA-D34.
 47. Amethod for producing a transgenic Poaceae plant comprising: (a)transforming a cell of a Poaceae explant with an expression constructcomprising a heterologous gene of interest; and (i) a nucleotidesequence encoding a WUS/WOX homeobox polypeptide; and (ii) a nucleotidesequence encoding a Babyboom (BBM) polypeptide or an Ovule DevelopmentProtein 2 (ODP2) polypeptide; and (b) allowing expression of theexpression construct of (a) in each transformed cell for about 0 toabout 7 days or for about 0 to about 14 days after initiation oftransforming the cell, wherein a somatic embryo is formed within about 0to about 7 days or within about 0 to about 14 days after initiation oftransforming the cell; and (c) germinating the somatic embryo of (b) forabout 14 to about 60 days to form a plantlet; and (d) allowing theplantlet of (c) to grow into a Poaceae plant.
 48. The method of claim47, wherein germinating comprises transferring the somatic embryo to amaturation medium and forming the transgenic Poaceae plant.
 49. Themethod of claim 47, wherein the expression construct further comprises anucleotide sequence encoding a site-specific recombinase selected fromFLP, Cre, SSV1, lambda Int, phi C31 Int, HK022, R, Gin, Tn1721, CinH,ParA, Tn5053, Bxb1, TP907-1, or U153.
 50. The method of claim 49,wherein the nucleotide sequence encoding a site-specific recombinase isoperably linked to a constitutive promoter, an inducible promoter, or adevelopmentally regulated promoter.
 51. The method of claim 47, wherein(c) is performed in the presence of exogenous cytokinin.
 52. The methodof claim 47, wherein the nucleotide sequence encoding the WUS/WOXhomeobox polypeptide and the nucleotide sequence encoding a polypeptidecomprising the BBM polypeptide or the ODP2 polypeptide is operablylinked to an inducible promoter, a developmentally regulated promoter,or a constitutive promoter.
 53. The method of claim 50, wherein theconstitutive promoter is selected from UBI, LLDAV, EVCV, DMMV, BSV (AY)PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO (ALT1), USB1ZM PRO,ZM-GOS2 PRO, ZM-H1B PRO (1.2 KB), IN2-2, NOS, the -135 version of 35S,or ZM-ADF PRO (ALT2); the inducible promoter is selected from AXIG1,DR5, XVE, GLB1, OLE, LTP2, HSP17.7, HSP26, HSP18A, or promotersactivated by tetracycline, ethamethsulfuron or chlorsulfuron; and thedevelopmentally regulated promoter is selected from PLTP, PLTP1, PLTP2,PLTP3, LGL, LEA-14A, or LEA-D34.
 54. The method of claim 52, wherein theconstitutive promoter is selected from UBI, LLDAV, EVCV, DMMV, BSV(AY)PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO (ALT1), USB1ZM PRO,ZM-GOS2 PRO, ZM-H1B PRO (1.2 KB), IN2-2, NOS, the -135 version of 35S,or ZM-ADF PRO (ALT2); the inducible promoter is selected from AXIG1,DR5, XVE, GLB1, OLE, LTP2, HSP17.7, HSP26, HSP18A, or promotersactivated by tetracycline, ethamethsulfuron or chlorsulfuron; and thedevelopmentally regulated promoter is selected from PLTP, PLTP1, PLTP2,PLTP3, SDR, LGL, LEA-14A, or LEA-D34.
 55. The method of claim 31,wherein the WUS/WOX homeobox polypeptide comprises the amino acidsequence of any of SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16, or wherein theWUS/WOX homeobox polypeptide is encoded by the nucleotide sequence ofany of SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15, and wherein the BBMpolypeptide or the Ovule Development Protein 2 (ODP2) polypeptidecomprises the amino acid sequence of any of SEQ ID NO: 18, 20, 63, 65,or 67, or wherein the BBM polypeptide or the Ovule Development Protein 2(ODP2) polypeptide is encoded by the nucleotide sequence of any of SEQID NO: 17, 19, 21, 62, 64, 66, or
 68. 56. The method of claim 47,wherein the WUS/WOX homeobox polypeptide comprises the amino acidsequence of any of SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16, or wherein theWUS/WOX homeobox polypeptide is encoded by the nucleotide sequence ofany of SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15, and wherein the BBMpolypeptide or the Ovule Development Protein 2 (ODP2) polypeptidecomprises the amino acid sequence of any of SEQ ID NO: 18, 20, 63, 65,or 67, or wherein the BBM polypeptide or the Ovule Development Protein 2(ODP2) polypeptide is encoded by the nucleotide sequence of any of SEQID NO: 17, 19, 21, 62, 64, 66, or
 68. 57. The method of claim 34,wherein the nucleotide sequence encoding the WUS/WOX homeoboxpolypeptide and the nucleotide sequence encoding the BBM polypeptide orthe ODP2 polypeptide is excised.
 58. The method of claim 42, wherein thenucleotide sequence encoding the WUS/WOX homeobox polypeptide and thenucleotide sequence encoding the BBM polypeptide or the ODP2 polypeptideis excised.
 59. The method of claim 50, wherein the nucleotide sequenceencoding the WUS/WOX homeobox polypeptide and the nucleotide sequenceencoding the BBM polypeptide or the ODP2 polypeptide is excised.